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Bacterial number and biomass in a meadow ecosystem
Zentralbl. Mikrobiol. 142 (1987), 559-568 VEB Gustav Fischer Verlag Jena [Department of Agricultural Microbiology, Academy of Agriculture, Poznan, Poland]
Bacterial N urn ber and Biomass in a Meadow Ecosystem HENRYK KASZUBIAK
and
MARIA MUSZYNSKA
With 4 Figures
Summary Bacterial numbers, determined under microscope in 1 g of the above ground plant material from a meadow, were usually of the range 109_1010 and many times greater than those determined by using the culture method, and similar to those found for soil. However, in the ecosystem the bacterial biomass of soil predominated quantitatively over the bacterial biomass of above ground plant material. During the vegetative season the latter was generally greater in living plant material than in decayed one. In the living plant material the bacterial biomass of leaves always predominated over that of stems. The bacterial community in the whole above ground plant rna· terial, compared with that in soil, was characterized by a relatively small proportion of endospores and dead cells, by higher dynamics, and by different reaction to slurry application.
Zusammenfassung Die Bakterienzahlen, die unter dem Mikroskop in 1 g des oberirdischen Pflanzenmaterials einer Wiese bestimmt wurden, lagen gewiihnlich im Bereich von 109 bis 1010 und waren mehrfach hiiher als jene, die mit der Ztichtungsmethode bestimmt worden waren, und denen l1hnlich, die im Boden gefunden wurden. J edoch dominiert die bakterielle Biomasse des Bodens im Okosystem quanti· tativ tiber die des oberirdischen Pflanzenmaterials. Wl1hrend der Vegetationsperiode war die Bakterienbiomasse des oberirdischen Pflanzenmaterials generell griiJ3er in lebendem Pflanzen· material hiiher als in totem. Die Bakterienbiomasse im lebenden Pflanzenmaterial war in Bll1ttern stets griiJ3er als in Stengeln. 1m Vergleich mit der Bakteriengemeinschaft im Boden war die Bak· teriengemeinschaft im Pflanzenmaterial charakterisiert durch einen relativ kleinen Prozentsatz von Endosporen und toten Zellen, durch griiJ3ere zahlenmiWige Dynamik und eine andere Re· aktion auf Gtilleanwendung.
In the terrestrial ecosystems bacteria are both in and above the soil where they inhabit mostly plants. The occurrence of bacteria in both environments are usually investigated independently, using different methods with different aims. Numerous data concerning bacterial number and biomass, obtained by means of the direct method, were collected for soil, while the investigations on the above ground occurrence of bacteria on plant shoots were often phytopathologic ally oriented. If the total bacterial number was determined in the same material it was always done using the culture method which does not reveal the whole community of these organisms, hence does not allow measuring its biomass. In the following work the authors aimed at a compaJison of bacterial numbers and their biomass concentrations in the whole above ground plant material and in the soil under the conditions of application of fertilizers and slurry dressing. Then we dealt with the distribution of bacteria in this plant material, limiting our considerations to the case of the mineral dressing of the meadow only. The material was classified in decayed plant material, including standing dead and litter, and into living plants. 39*
560
H. KASZUBIAK and M. MUSZYNSKA
In the latter the occurrence of bacteria on leaves, including leaf pockets, and on stems with spikes was examined. We also tried to recognize the vitality of the bacteria of the above ground plant material, determining the percentage of living and dead cells as well as that of vegetative forms and endospores in the community.
Materials and methods Meadow and enrichment The investigations were carried out on a meadow belonging to the Experimental Station in Brody. The meadow was on boggy soil of pH = 6.8, containing 8.1 % organic C. The predominating grasses were Poa pratensi8, BromU8 inermi8, Poa triviali8, and Fe8tuca rubra. From the plots with different enrichment, the plot receiving a moderate mineral fertilization (200 kg Njha) and the intensively slurry-dressed one (400 kg Njha), applied to every grass regrowth, were selected. The detailed data concerning the meadow and its enrichment scheme were described earlier (KASZUBIAK and MU8ZYNSKA 1987). On both plots the examination was carried out in 1983, and on the mineral treated one examinations were continued in 1984.
Sampling and preparation of the samples for analyses Sampling were taken 6 times during the vegetative season: after every re-growth of the grasses up to grazing stage and at mowing time (Table 1). The material was collected along a diagonal line of the examined plots. Table 1. Times of analyses of the tested material Re-growth First
grazing stage mowing time
3 May 24 May
2 May 29 May
Second
grazing stage mowing time
5 July 25 July
10 July 26 July
Third
grazing stage mowing time
12 Sept. 28 Sept.
20 Sept. 9 Oct.
Representative soil samples consisted of 30-40 subsamples, taken from 30-cm top layer. Representative plant material samples contained the material collected in 6 places, 3 from every diagonal line. An area of each place was 0.25 m 2 • Analyses were done not later than 6 h after sampling. The tested material in 10 g portions were ground in 100 ml of 0.2 % Na4 P a0 7 in a homogenizer at 1,400 r.p.m. for 90 s. In the case of plant material, it was filtered through gauze. Sometimes, as pointed out in the results, the obtained suspensions were sonicated to increase bacterial desorption from the examined material. The desorption was carried out by a 22-kHz disintegrator at different ranges of vibrations and time.
Determinations 1. Count bacteria The bacteria were enumerated in the obtained suspensions, using two methods parallel: the culture method (plate-dilution-method) and the microscopic one. For the culture method the usefulness of two media were initially determined: the WALLACE and LOCHHEAD'S medium (1950), with soil extract as a sole carbon and nitrogen source, and the nitrate glucose medium, prepared according to WEST and LOCHHEAD (1940), supplied with 0.2 % casein acid hydrolysate according to KASZUBIAK and RATAJCZAK (1973).
Bacterial Number and Biomass in a Meadow Ecosystem
561
The soil bacteria colonies were slightly more numerous on the first medium, compared to those from the plant material on the second medium. Since the differences observed for each medium were not significant for the results' interpretation, this paper presents only those numerical data which were obtained by media optimum for a given material. Direct counting of the bacteria was carried out under a contrast-phase microscope with the stained and fixed preparations. The plant material suspensions were stained for 24 h with aniline blue according to JONES and MOLLISON (1948). For the soil suspension, preparations proved more useful by staining with erythrosine according to WJNOGRADSKI, though it did not increase cell detectability. All the determined bacteria numbers were calculated per 1 g dry wt. of the tested material. If it was needful to differentiate the living and dead bacterial cells under microscope, the fluorescent microscope was used with acridine orange as a fluorochrome (STRUGGER 1948). The fluorochrome concentration was determined separately for the soil and plant material. The optimum acridine orange concentration was such at which the cells of 24-h-Azotobacter culture fluoresced as living cells of an apple-green light after introducing them into the tested material.
2. Bacterial biomass The biomass concentration was calculated on the basis of the bacteria number, determined with contrast-phase microscope in the fixed preparations, assuming that the average volume of the bacterial cell was 0.22,um3 (KASZUBIAK et al. 1977), its specific weight 1.1 (ZVYAGlNTSEV and ROGACHEVSKIY 1973), and the water content in fresh weight 70 %.
Results 1. Bacterial number and biomass per weight unit of the tested material Fig. 1 and Table 2 present the data concerning bacterial number in 1 g of soil and plant material, not differentiated into its components. In the soil the numbers of units visible under the microscope (numbers of MVU) were in the range of 109 or 1010 , and those determined with the plate method (numbers of CFU) in the range of 107• In the plant material the bacteria were also abundant. The MVU numbers were in the same range like those in the soil, and the CFU ones even much greater, often in the range of lOS or even of 109 • Despite the kind of tested material the MVU numbers have shown less fluctuations during the vegetative season, but the fluctuations of both numbers were greater in the plant material than in the soil. In the soil the changes in the MVU and CFU numbers were not clearly directed. In the plant material the CFU numbers changed in a similar way, whereas the MVU numbers decreased considerably by the end of the 1984 vegetative season, during which after the second mowing grasses grew more slowly than in 1983; due to this the third mowing was done in October. Slurry, despite its significant bacterial number and high concentration of nutrient, did not significantly rise the bacterial number in the tested material. In the soil dressed with slurry the increments in the CFU number were noted only occasionally. Generally, the tendency towards slight decrease was observed_ The MVU number always fell considerably and reached 61 %. If the increments of the bacterial number were observed at all in the plant material, they were noted mainly under the microscope. However, independent of the tested material and the direction of the produced changes, slurry dressing of the meadow intensified the dynamics of changes in the bacterial number. Counting the bacteria under the microscope, it was found that in the soil they occurred mostly on its particles where they created microcolonies. On the plants they
562
H. KASZUBIAK and M. MUSZYNSKA
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PLATE COUNT 10
PLANT MATERIAL
1984
1983
1983
9
B
a
I
b
a
II
a
b
III
a
b ill
b
MICROSCOPIC COUNT PLANT MATERIAL
SOIL
1984
1983
19B3
10
9
B
a
I
b
a
II
b
a
ill
0- NPK • - Slurry I II III - Regrowths
b
a
I
b
a
II
b
a
b
ab ab ab I II ill
ill
a - Grazing stage b - Mowing time
Fig.!. Bacterial number in the weight unit of soil and of above-ground plant material. 0 - NPK; • - slurry; I, II, III - re-growths; a - grazing stage; b - mowing time.
Table 2. Variation coefficient (V) for bacterial number in soil and in above ground plant material Microscopic count
Plate count
Experimental year
Tested material
Meadow enrichment
1983
Soil
NPK salts Slurry
24 82
18 26
Plant
NPK salts Slurry
80 242
35 39
Plant
NPK salts
95
69
1984
V
Bacterial Number and BiomaRs in a Meadow Ecosystem
563
PLATE COUNT LIVING AND DEAD PLANT MATERIAL
a
bob I II
a
m
LIVING LEAVES AND STEMS
b
I
a
b
a
I
a II
b III
MICROSCOPIC COUNT LIVING AND DEAD PLANT MATERIAL
11
a
I
b
a
II
b
a
III
LIVING LEAVES AND STEMS
b
a
b
I
a II
a
b III
Fig. 2. Bacterial number in the weight unit of various fractions of aboye-ground plant material (1984). -living plant material or living leayos; • - dead plant material or living stems; I, II, IIIre-growths; a - grazing stage; b - mowing time.
o
were found in clusters, located along the nerves and around trichomes, which could be one of the reasons for the underestimated CFU numbers as compared to the MVU ones. During the attempts to disintegrate these clusters and desorb the bacteria by means of sonication at initially assumed optimum values of vibration amplitudes and time (14,um and 1 min, respectively), the CFU numbers for the soil could be increased only 2.4 times and for the plant material 1.3 times. At the vibration amplitude higher than the optimum value and at prolonged time of sonication, the CFU number for the plant material fell rapidly, while for the soil this occurred gradually. Both the microscopic and culture method not always produced compatible information concerning bacteria distribution in the plant material (Fig. 2). By means of the former method it was generally found that per 1 g of living plant there occurred
564
H. KASZUBIAK and M. MUSZYNSKA
Table 3. Bacterial biomass in the weight unit of soil and of above ground plant material (mg/g dry wt.)1) Tested material
Experimental year
Soil
1983
0.74-1.23 (0.32-0.70)
1.04 (0.59)
Plant material as a whole
1983 1984
0.34--0.82 (0.35-1.12) 0.14-1.55
0.50 (0.72) 0.85
1984
0.07-0.58
0.27
1984
0.15-2.13
1.16
1984 1984
0.17-3.38 0.03-0.80
1.60 0.37
Decayed plant material as a whole Living plant material as a whole Living leaves Living stems
Range
Average
1) Data for NPK fertilization without brackets, data for slurry dressing in brackets
more bacteria than per 1 g of decayed plants, whereas by means of the latter method the opposite relation was most often found. Moreover, in the living plant material under the microscope, more bacteria were always found in 1 g of leaves than in 1 g of stems, what was confirmed by the culture method only at certain times of analysing; in most cases the numerical relations were opposite. The bacterial biomass concentration in the tested material is given in Table 3, considering only the ranges of fluctuations and average values. In the biomass calculations it was assumed that it was directly proportional to the MVU number, and thus the numerical relations between each biomass concentrations were the same as between the respective MVU numbers. 2. Bacterial biomass concentration per area unit of the meadow The 30-cm top layer of 1 m 2 area contained 450 kg soil, thus, from the data presented in Fig. 1, we calculated that its bacterial biomass corresponded to lOO g. The amount of the whole above ground plant material on this area was about several hundreds g. Therefore, in contradistinction to the data, obtained from calculation per weight unit, the bacterial biomass in the above ground plant material was in the ecosystem many times smaller than that in the soil and corresponded to lO or hundreds of milligrams per 1 m 2 (Fig. 3). In every grass re-glOwth it was greater during mowing time than at grazing stage and fell at the end of vegetative season, as observed in 1984. Comparison of Figs. 3 and 1 shows that the changes, occurring during every grass re-growth, were mainly caused by different amounts of the plant material on the meadow, while the changes taking place at the end of vegetative season were due to decrease in hacterial nnmber in weight unit of this lllateriai. In the above ground bacterial biomass of the ecosystem predominated organisms occurring on the living plants. This was marked even during the spring grass regrowth up to grazing stage, when dead plant material, remaining after winter predominated quantitatively over green grass mass (Fig. 4). Later, when living plants predominated in the plant material, the domination of their bacteria was even more pronounced. During the second re-growth the bacteria, colonizing liver plants,
Bacterial Number and Biomass in a Meadow Ecosystem
BACTERIAL BavtASS
PLANT MATERIAL
.
1983
565
1984
1983
1984 600
Fig. 3. Total above· ground material and its bacterial biomass on the meadow. 0 - NPK; • - slurry; I, II, III, - re·growths; a - grazing stage; b - mowing time.
constituted over 90 % of the above ground bacterial biomass. Only during the third mowing, when dead plant material accumulated again on the meadow, its bacterial biomass predominated over that of bacteria of living plants. The bacteria of living plants were distributed in the ecosystem mainly on leaves and constituted usually 94-98 % of the above ground bacterial biomass, and only during the first mowing 64 %, since at that time the mass of stems was greater than that of leaves. 3. Vitality of bacterial communities of the tested material Examination of bacterial cells in fixed microscopic preparations did not provide information concerning their vitality. To determine this, the suspensions of homogenized material were stained with acridine orange and observed under fluoresent microscope. This method proved useful only in case of soil suspensions in which living cells, fluoresent of an apple-green light, constituted 90-95 % of visible bacteria. In
PLANT MATERIAL ......
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Fig. 4. Living above-ground plant material and its bacterial biomass (1984). total; • - living leaves; I, II, III - re-growths; a - grazing time; b -
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ab ill
mowing time
566
H.
KASZUBIAK
and
M. MUSZYNSKA
the microscopic pictures of the plant material the bacteria were seen mainly on the background of this material and were poorly visible, since it fluoresced yellow. After separating by means of filtering through cotton, 97-99 % of the bacteria, not absorbed on plant tissues, fluoresced as living cells. The CFU numbers determined for this filtrate corresponded to 23-42 % of the total bacterial number, determined with the microscope. Table 4. Percentage of endospores in bacterial number, determined by plate count on the ffi81dow in the soil and in the above ground plant material as a whole!) Meadow maturity
NPK salts
Slurry dressing
Soil
Soil
Plant material
First
Grazing stage Mowing time
3.76 4.40
0.31 0.01
3.30 4.42
0.01 0.01
Second
Grazing stage Mowing time
5.48 4.55
0.14 18.17
2.69 3.69
0.11 7.11
Third
Grazing stage Mowing time
1.13 5.44
0.02 0.02
1.02 5.01
0.01 0.02
Re·growth
Plant material
1) Data from 1983y
In the investigations on the bacterial endospores occurrence, it was found that in the plant material their CFU number of the total CFU ratio was lower than in the soil (Table 4). An exception showed the second mowing in which the percentage of endospores was higher for the plant material than for the soil. Howevm, this was observed during great drought, when the plants were on the threshold of wilting and covered with dust, carried by wind from the fields.
Discussion As could be expected (KACZMAREK et al. 1973), at least hundred times more bacteria were found in the soil by means of the microscope than with the culture method, in which the bacterial clusters were the same calculation units as single cells, and the used media were always selective. In the above ground plant material, too, much more bacteria were detected, using the microscope instead of the culture method, confirming the results obtained by TESARovA (1983). It was proved that the differences, noted for the plant material, to a less extent resulted from the selectively of the medium used, but rather from the adsorption of the bacterial cells on plant tissues in centes. Attempts to obtain highm values by means of the culture method by dispersing these clusters and desorption of the bacteria from the plant surface had small effectiveness, probably resulting from high sensitivity of these bacterial communities, mostly composed of Gram-negative cells, to the destructive effect of ultrasound. Our experiments differed from those by TEsARovA with respect to the subject matter, scope, and methodology. In TEsARovA's investigation the plant material, derived from a mountain meadow of a small productivity, was overgrown with N ardus stricta. The bacteria were not examined under microscope in homogenized plant material, but in suspensions obtained by means of rinsing the plants with water, without taking into account the phenomenon of strong sorption, which was also pointed out by GUZEV et al. (1984).
Bacterial Number and Biomass in a Meadow Ecosystem
567
Neglecting the differences of absolute numerical values, there still exist differences between ours' and TEsARovA's bacterial numbers and biomasses per weight unit of living and decayed plants. TEsARovA always found more bacteria in the latter, whereas in our experiments higher colonization of the former was observed, when the bacteria were counted under microscope. Detailed observations during an additional, so far not described, experiment showed that the bacteria on the surface of decayed piant tissues, in contradistinction to living tissues, were dispersed and weakly adsorbed, since their majority occurred freely in the suspensions of tested material. Therefore, when the suspensions of this material were made according to TESAROVA, the obtained result stimulated more numerous appearance of bacteria on decayed plants than on living ones. Greater occurrence of bacteria on leaves than on stems, and also the drop in their number by the end of vegetative season, indicates that the bacterial biomass of the above ground parts of the plants consisted mostly of the cells assimilating plant exudates, whose concentration was limited by the intensity of photosynthesis. Although in the above ground plant material occurred only a small portion of the total bacterial biomass of the ecosystem, it was, however, a component of a relatively high activity. As compared to the soil bacterial biomass, there was a greater proportion of living cells, but less of endospores. Its dynamics was higher, as well as probably its renewing. Epiphytic microflora, as compared to that of soil, is more exposed to various stresses repeated even at day intervals. During a day, due to evaporation of the night dew and to the activity of ultraviolet rays, part of the bacterial community dies to renew itself after disappearance of this stress. The results concerning the effect of slurry on the bacteria in a meadow ecosystem indicate that the changes in the bacterial number or biomass concentration, taking place on the plant surface, may occur under the influence of a given factor, different from those acting in the soil. They also confirm our earlier findings (KASZUBIAK et al. 1983; MUSZYNSKA and KASZUBIAK 1985) concerning the negative effect of slurry, used in high doses on the bacterial biomass concentration in soil, thus proving the universality of this phenomenon. Acknowledgement This work was carried out under the project MR II 23, coordinated by the Department of Agrobiology and Forestry, Polish Academy of Sciences.
References GUZEV, W. S., KULICHEVSKAYA, 1. S., and ZVYAGINTSEV, D. G.: Skaninguyushchaya elektronnaya mikroskopiya pri izucheniy wzaimodieystviya mikroorganizmov s rastieniyami. In: Mikro· organizmy kak komponenty biogeocenoza (E. N. MISHUSTIN, ed.) Moscow 1984, 92-107. JONES, P. C. T., and MOLLISON, J. N.: A technique for the quantitative estimation of soil microorganisms. J. Gen. Microbiol. 2 (1948), 54-69. KACZMAREK, W., KASZUBIAK, R., and GUZEK, R.: Comparison of changes in the number of microorganisms in the soil by the plate and microscopic procedures. Pol. J. Soil Sci. 6 (1973), 133 to 139. KASZUBIAK, R., DURSKA, G., KACZMAREK, W., and FILODA, G.: Effect of slurry on microorganisms and chemical properties of soil. Zbl. Mikrobiol. 138 (1983), 501-509. - KACZMAREK, W., and Plj)DZIWILK, Z.: Comparison of different methods fo estimating thes productivity of microorganisms in soil. Ekol. Pol. 25 (1977), 289-296. - and MUSZYNSKA, M.: Bacteria of meadow sward and the effect of slurry dressing on their community. Zentralbl. Mikrobiol. 142 (1987), 13-21. - and RATAJCZAK, R.: Wplyw bromoxynilu na drobnoustroje w ryzosferze j~czmienia. PTPN, Prace Komisji Nauk Roln. i Komisji Nouk Lesnych 35 (1973), 185-189.
568
H. KASZUBIAK and M. MUSZYNSKA, Bacterial Number and Biomass in a Meadow
MUSZYNSKA, M., and KASZUBIAK, H.: The influence of slurry on the bacterial biomass in incubated soil samples. Zbl. Mikrobiol. 140 (1985), 277-282. STRUGGER, S.: A fluorescent microscope examination of bacteria in soil. Can. J. Res., Sec. C, 26 (1948),188-193. TEsARovA, M.: Bakterienbiomasse in verschieden bewirtschafteten Wiesen-Okosystemen. Zbl. Mikrobiol. 138 (1983), 179-186. WALLACE, R. H., and LOCHHEAD, A. G.: Qualitative studies of soil microorganisms. IX. Amino acid requirements of rhizosphere bacteria. Can. J. Res., Sec. C 28 (1950), 1-6. WEST, P. M., and LOCHHEAD, A. G.: Quantitative studies of soil microorganisms. IV. The rhizosphere in relation to the nutritive requirements of soil bacteria. Can. J. Res. 18 (1940),129-136. ZVYAGINTSEV, D. G., and ROGACHEVSKIY, L. M.: Plotnost (udielniy vies) kletok mikroorganizmov. Mikrobiologiya 42 (1973), 892-898. Authors' address: Prof. Dr. HENRYK KASZUBIAK and Dr. MARIA MUSZYNSKA, Department of Agricultural Microbiology, Academy of Agriculture, W olynska 35. 60-637 Poznan. Poland.
Bacterial N urn ber and Biomass in a Meadow Ecosystem HENRYK KASZUBIAK
and
MARIA MUSZYNSKA
With 4 Figures
Summary Bacterial numbers, determined under microscope in 1 g of the above ground plant material from a meadow, were usually of the range 109_1010 and many times greater than those determined by using the culture method, and similar to those found for soil. However, in the ecosystem the bacterial biomass of soil predominated quantitatively over the bacterial biomass of above ground plant material. During the vegetative season the latter was generally greater in living plant material than in decayed one. In the living plant material the bacterial biomass of leaves always predominated over that of stems. The bacterial community in the whole above ground plant rna· terial, compared with that in soil, was characterized by a relatively small proportion of endospores and dead cells, by higher dynamics, and by different reaction to slurry application.
Zusammenfassung Die Bakterienzahlen, die unter dem Mikroskop in 1 g des oberirdischen Pflanzenmaterials einer Wiese bestimmt wurden, lagen gewiihnlich im Bereich von 109 bis 1010 und waren mehrfach hiiher als jene, die mit der Ztichtungsmethode bestimmt worden waren, und denen l1hnlich, die im Boden gefunden wurden. J edoch dominiert die bakterielle Biomasse des Bodens im Okosystem quanti· tativ tiber die des oberirdischen Pflanzenmaterials. Wl1hrend der Vegetationsperiode war die Bakterienbiomasse des oberirdischen Pflanzenmaterials generell griiJ3er in lebendem Pflanzen· material hiiher als in totem. Die Bakterienbiomasse im lebenden Pflanzenmaterial war in Bll1ttern stets griiJ3er als in Stengeln. 1m Vergleich mit der Bakteriengemeinschaft im Boden war die Bak· teriengemeinschaft im Pflanzenmaterial charakterisiert durch einen relativ kleinen Prozentsatz von Endosporen und toten Zellen, durch griiJ3ere zahlenmiWige Dynamik und eine andere Re· aktion auf Gtilleanwendung.
In the terrestrial ecosystems bacteria are both in and above the soil where they inhabit mostly plants. The occurrence of bacteria in both environments are usually investigated independently, using different methods with different aims. Numerous data concerning bacterial number and biomass, obtained by means of the direct method, were collected for soil, while the investigations on the above ground occurrence of bacteria on plant shoots were often phytopathologic ally oriented. If the total bacterial number was determined in the same material it was always done using the culture method which does not reveal the whole community of these organisms, hence does not allow measuring its biomass. In the following work the authors aimed at a compaJison of bacterial numbers and their biomass concentrations in the whole above ground plant material and in the soil under the conditions of application of fertilizers and slurry dressing. Then we dealt with the distribution of bacteria in this plant material, limiting our considerations to the case of the mineral dressing of the meadow only. The material was classified in decayed plant material, including standing dead and litter, and into living plants. 39*
560
H. KASZUBIAK and M. MUSZYNSKA
In the latter the occurrence of bacteria on leaves, including leaf pockets, and on stems with spikes was examined. We also tried to recognize the vitality of the bacteria of the above ground plant material, determining the percentage of living and dead cells as well as that of vegetative forms and endospores in the community.
Materials and methods Meadow and enrichment The investigations were carried out on a meadow belonging to the Experimental Station in Brody. The meadow was on boggy soil of pH = 6.8, containing 8.1 % organic C. The predominating grasses were Poa pratensi8, BromU8 inermi8, Poa triviali8, and Fe8tuca rubra. From the plots with different enrichment, the plot receiving a moderate mineral fertilization (200 kg Njha) and the intensively slurry-dressed one (400 kg Njha), applied to every grass regrowth, were selected. The detailed data concerning the meadow and its enrichment scheme were described earlier (KASZUBIAK and MU8ZYNSKA 1987). On both plots the examination was carried out in 1983, and on the mineral treated one examinations were continued in 1984.
Sampling and preparation of the samples for analyses Sampling were taken 6 times during the vegetative season: after every re-growth of the grasses up to grazing stage and at mowing time (Table 1). The material was collected along a diagonal line of the examined plots. Table 1. Times of analyses of the tested material Re-growth First
grazing stage mowing time
3 May 24 May
2 May 29 May
Second
grazing stage mowing time
5 July 25 July
10 July 26 July
Third
grazing stage mowing time
12 Sept. 28 Sept.
20 Sept. 9 Oct.
Representative soil samples consisted of 30-40 subsamples, taken from 30-cm top layer. Representative plant material samples contained the material collected in 6 places, 3 from every diagonal line. An area of each place was 0.25 m 2 • Analyses were done not later than 6 h after sampling. The tested material in 10 g portions were ground in 100 ml of 0.2 % Na4 P a0 7 in a homogenizer at 1,400 r.p.m. for 90 s. In the case of plant material, it was filtered through gauze. Sometimes, as pointed out in the results, the obtained suspensions were sonicated to increase bacterial desorption from the examined material. The desorption was carried out by a 22-kHz disintegrator at different ranges of vibrations and time.
Determinations 1. Count bacteria The bacteria were enumerated in the obtained suspensions, using two methods parallel: the culture method (plate-dilution-method) and the microscopic one. For the culture method the usefulness of two media were initially determined: the WALLACE and LOCHHEAD'S medium (1950), with soil extract as a sole carbon and nitrogen source, and the nitrate glucose medium, prepared according to WEST and LOCHHEAD (1940), supplied with 0.2 % casein acid hydrolysate according to KASZUBIAK and RATAJCZAK (1973).
Bacterial Number and Biomass in a Meadow Ecosystem
561
The soil bacteria colonies were slightly more numerous on the first medium, compared to those from the plant material on the second medium. Since the differences observed for each medium were not significant for the results' interpretation, this paper presents only those numerical data which were obtained by media optimum for a given material. Direct counting of the bacteria was carried out under a contrast-phase microscope with the stained and fixed preparations. The plant material suspensions were stained for 24 h with aniline blue according to JONES and MOLLISON (1948). For the soil suspension, preparations proved more useful by staining with erythrosine according to WJNOGRADSKI, though it did not increase cell detectability. All the determined bacteria numbers were calculated per 1 g dry wt. of the tested material. If it was needful to differentiate the living and dead bacterial cells under microscope, the fluorescent microscope was used with acridine orange as a fluorochrome (STRUGGER 1948). The fluorochrome concentration was determined separately for the soil and plant material. The optimum acridine orange concentration was such at which the cells of 24-h-Azotobacter culture fluoresced as living cells of an apple-green light after introducing them into the tested material.
2. Bacterial biomass The biomass concentration was calculated on the basis of the bacteria number, determined with contrast-phase microscope in the fixed preparations, assuming that the average volume of the bacterial cell was 0.22,um3 (KASZUBIAK et al. 1977), its specific weight 1.1 (ZVYAGlNTSEV and ROGACHEVSKIY 1973), and the water content in fresh weight 70 %.
Results 1. Bacterial number and biomass per weight unit of the tested material Fig. 1 and Table 2 present the data concerning bacterial number in 1 g of soil and plant material, not differentiated into its components. In the soil the numbers of units visible under the microscope (numbers of MVU) were in the range of 109 or 1010 , and those determined with the plate method (numbers of CFU) in the range of 107• In the plant material the bacteria were also abundant. The MVU numbers were in the same range like those in the soil, and the CFU ones even much greater, often in the range of lOS or even of 109 • Despite the kind of tested material the MVU numbers have shown less fluctuations during the vegetative season, but the fluctuations of both numbers were greater in the plant material than in the soil. In the soil the changes in the MVU and CFU numbers were not clearly directed. In the plant material the CFU numbers changed in a similar way, whereas the MVU numbers decreased considerably by the end of the 1984 vegetative season, during which after the second mowing grasses grew more slowly than in 1983; due to this the third mowing was done in October. Slurry, despite its significant bacterial number and high concentration of nutrient, did not significantly rise the bacterial number in the tested material. In the soil dressed with slurry the increments in the CFU number were noted only occasionally. Generally, the tendency towards slight decrease was observed_ The MVU number always fell considerably and reached 61 %. If the increments of the bacterial number were observed at all in the plant material, they were noted mainly under the microscope. However, independent of the tested material and the direction of the produced changes, slurry dressing of the meadow intensified the dynamics of changes in the bacterial number. Counting the bacteria under the microscope, it was found that in the soil they occurred mostly on its particles where they created microcolonies. On the plants they
562
H. KASZUBIAK and M. MUSZYNSKA
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PLATE COUNT 10
PLANT MATERIAL
1984
1983
1983
9
B
a
I
b
a
II
a
b
III
a
b ill
b
MICROSCOPIC COUNT PLANT MATERIAL
SOIL
1984
1983
19B3
10
9
B
a
I
b
a
II
b
a
ill
0- NPK • - Slurry I II III - Regrowths
b
a
I
b
a
II
b
a
b
ab ab ab I II ill
ill
a - Grazing stage b - Mowing time
Fig.!. Bacterial number in the weight unit of soil and of above-ground plant material. 0 - NPK; • - slurry; I, II, III - re-growths; a - grazing stage; b - mowing time.
Table 2. Variation coefficient (V) for bacterial number in soil and in above ground plant material Microscopic count
Plate count
Experimental year
Tested material
Meadow enrichment
1983
Soil
NPK salts Slurry
24 82
18 26
Plant
NPK salts Slurry
80 242
35 39
Plant
NPK salts
95
69
1984
V
Bacterial Number and BiomaRs in a Meadow Ecosystem
563
PLATE COUNT LIVING AND DEAD PLANT MATERIAL
a
bob I II
a
m
LIVING LEAVES AND STEMS
b
I
a
b
a
I
a II
b III
MICROSCOPIC COUNT LIVING AND DEAD PLANT MATERIAL
11
a
I
b
a
II
b
a
III
LIVING LEAVES AND STEMS
b
a
b
I
a II
a
b III
Fig. 2. Bacterial number in the weight unit of various fractions of aboye-ground plant material (1984). -living plant material or living leayos; • - dead plant material or living stems; I, II, IIIre-growths; a - grazing stage; b - mowing time.
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were found in clusters, located along the nerves and around trichomes, which could be one of the reasons for the underestimated CFU numbers as compared to the MVU ones. During the attempts to disintegrate these clusters and desorb the bacteria by means of sonication at initially assumed optimum values of vibration amplitudes and time (14,um and 1 min, respectively), the CFU numbers for the soil could be increased only 2.4 times and for the plant material 1.3 times. At the vibration amplitude higher than the optimum value and at prolonged time of sonication, the CFU number for the plant material fell rapidly, while for the soil this occurred gradually. Both the microscopic and culture method not always produced compatible information concerning bacteria distribution in the plant material (Fig. 2). By means of the former method it was generally found that per 1 g of living plant there occurred
564
H. KASZUBIAK and M. MUSZYNSKA
Table 3. Bacterial biomass in the weight unit of soil and of above ground plant material (mg/g dry wt.)1) Tested material
Experimental year
Soil
1983
0.74-1.23 (0.32-0.70)
1.04 (0.59)
Plant material as a whole
1983 1984
0.34--0.82 (0.35-1.12) 0.14-1.55
0.50 (0.72) 0.85
1984
0.07-0.58
0.27
1984
0.15-2.13
1.16
1984 1984
0.17-3.38 0.03-0.80
1.60 0.37
Decayed plant material as a whole Living plant material as a whole Living leaves Living stems
Range
Average
1) Data for NPK fertilization without brackets, data for slurry dressing in brackets
more bacteria than per 1 g of decayed plants, whereas by means of the latter method the opposite relation was most often found. Moreover, in the living plant material under the microscope, more bacteria were always found in 1 g of leaves than in 1 g of stems, what was confirmed by the culture method only at certain times of analysing; in most cases the numerical relations were opposite. The bacterial biomass concentration in the tested material is given in Table 3, considering only the ranges of fluctuations and average values. In the biomass calculations it was assumed that it was directly proportional to the MVU number, and thus the numerical relations between each biomass concentrations were the same as between the respective MVU numbers. 2. Bacterial biomass concentration per area unit of the meadow The 30-cm top layer of 1 m 2 area contained 450 kg soil, thus, from the data presented in Fig. 1, we calculated that its bacterial biomass corresponded to lOO g. The amount of the whole above ground plant material on this area was about several hundreds g. Therefore, in contradistinction to the data, obtained from calculation per weight unit, the bacterial biomass in the above ground plant material was in the ecosystem many times smaller than that in the soil and corresponded to lO or hundreds of milligrams per 1 m 2 (Fig. 3). In every grass re-glOwth it was greater during mowing time than at grazing stage and fell at the end of vegetative season, as observed in 1984. Comparison of Figs. 3 and 1 shows that the changes, occurring during every grass re-growth, were mainly caused by different amounts of the plant material on the meadow, while the changes taking place at the end of vegetative season were due to decrease in hacterial nnmber in weight unit of this lllateriai. In the above ground bacterial biomass of the ecosystem predominated organisms occurring on the living plants. This was marked even during the spring grass regrowth up to grazing stage, when dead plant material, remaining after winter predominated quantitatively over green grass mass (Fig. 4). Later, when living plants predominated in the plant material, the domination of their bacteria was even more pronounced. During the second re-growth the bacteria, colonizing liver plants,
Bacterial Number and Biomass in a Meadow Ecosystem
BACTERIAL BavtASS
PLANT MATERIAL
.
1983
565
1984
1983
1984 600
Fig. 3. Total above· ground material and its bacterial biomass on the meadow. 0 - NPK; • - slurry; I, II, III, - re·growths; a - grazing stage; b - mowing time.
constituted over 90 % of the above ground bacterial biomass. Only during the third mowing, when dead plant material accumulated again on the meadow, its bacterial biomass predominated over that of bacteria of living plants. The bacteria of living plants were distributed in the ecosystem mainly on leaves and constituted usually 94-98 % of the above ground bacterial biomass, and only during the first mowing 64 %, since at that time the mass of stems was greater than that of leaves. 3. Vitality of bacterial communities of the tested material Examination of bacterial cells in fixed microscopic preparations did not provide information concerning their vitality. To determine this, the suspensions of homogenized material were stained with acridine orange and observed under fluoresent microscope. This method proved useful only in case of soil suspensions in which living cells, fluoresent of an apple-green light, constituted 90-95 % of visible bacteria. In
PLANT MATERIAL ......
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mowing time
566
H.
KASZUBIAK
and
M. MUSZYNSKA
the microscopic pictures of the plant material the bacteria were seen mainly on the background of this material and were poorly visible, since it fluoresced yellow. After separating by means of filtering through cotton, 97-99 % of the bacteria, not absorbed on plant tissues, fluoresced as living cells. The CFU numbers determined for this filtrate corresponded to 23-42 % of the total bacterial number, determined with the microscope. Table 4. Percentage of endospores in bacterial number, determined by plate count on the ffi81dow in the soil and in the above ground plant material as a whole!) Meadow maturity
NPK salts
Slurry dressing
Soil
Soil
Plant material
First
Grazing stage Mowing time
3.76 4.40
0.31 0.01
3.30 4.42
0.01 0.01
Second
Grazing stage Mowing time
5.48 4.55
0.14 18.17
2.69 3.69
0.11 7.11
Third
Grazing stage Mowing time
1.13 5.44
0.02 0.02
1.02 5.01
0.01 0.02
Re·growth
Plant material
1) Data from 1983y
In the investigations on the bacterial endospores occurrence, it was found that in the plant material their CFU number of the total CFU ratio was lower than in the soil (Table 4). An exception showed the second mowing in which the percentage of endospores was higher for the plant material than for the soil. Howevm, this was observed during great drought, when the plants were on the threshold of wilting and covered with dust, carried by wind from the fields.
Discussion As could be expected (KACZMAREK et al. 1973), at least hundred times more bacteria were found in the soil by means of the microscope than with the culture method, in which the bacterial clusters were the same calculation units as single cells, and the used media were always selective. In the above ground plant material, too, much more bacteria were detected, using the microscope instead of the culture method, confirming the results obtained by TESARovA (1983). It was proved that the differences, noted for the plant material, to a less extent resulted from the selectively of the medium used, but rather from the adsorption of the bacterial cells on plant tissues in centes. Attempts to obtain highm values by means of the culture method by dispersing these clusters and desorption of the bacteria from the plant surface had small effectiveness, probably resulting from high sensitivity of these bacterial communities, mostly composed of Gram-negative cells, to the destructive effect of ultrasound. Our experiments differed from those by TEsARovA with respect to the subject matter, scope, and methodology. In TEsARovA's investigation the plant material, derived from a mountain meadow of a small productivity, was overgrown with N ardus stricta. The bacteria were not examined under microscope in homogenized plant material, but in suspensions obtained by means of rinsing the plants with water, without taking into account the phenomenon of strong sorption, which was also pointed out by GUZEV et al. (1984).
Bacterial Number and Biomass in a Meadow Ecosystem
567
Neglecting the differences of absolute numerical values, there still exist differences between ours' and TEsARovA's bacterial numbers and biomasses per weight unit of living and decayed plants. TEsARovA always found more bacteria in the latter, whereas in our experiments higher colonization of the former was observed, when the bacteria were counted under microscope. Detailed observations during an additional, so far not described, experiment showed that the bacteria on the surface of decayed piant tissues, in contradistinction to living tissues, were dispersed and weakly adsorbed, since their majority occurred freely in the suspensions of tested material. Therefore, when the suspensions of this material were made according to TESAROVA, the obtained result stimulated more numerous appearance of bacteria on decayed plants than on living ones. Greater occurrence of bacteria on leaves than on stems, and also the drop in their number by the end of vegetative season, indicates that the bacterial biomass of the above ground parts of the plants consisted mostly of the cells assimilating plant exudates, whose concentration was limited by the intensity of photosynthesis. Although in the above ground plant material occurred only a small portion of the total bacterial biomass of the ecosystem, it was, however, a component of a relatively high activity. As compared to the soil bacterial biomass, there was a greater proportion of living cells, but less of endospores. Its dynamics was higher, as well as probably its renewing. Epiphytic microflora, as compared to that of soil, is more exposed to various stresses repeated even at day intervals. During a day, due to evaporation of the night dew and to the activity of ultraviolet rays, part of the bacterial community dies to renew itself after disappearance of this stress. The results concerning the effect of slurry on the bacteria in a meadow ecosystem indicate that the changes in the bacterial number or biomass concentration, taking place on the plant surface, may occur under the influence of a given factor, different from those acting in the soil. They also confirm our earlier findings (KASZUBIAK et al. 1983; MUSZYNSKA and KASZUBIAK 1985) concerning the negative effect of slurry, used in high doses on the bacterial biomass concentration in soil, thus proving the universality of this phenomenon. Acknowledgement This work was carried out under the project MR II 23, coordinated by the Department of Agrobiology and Forestry, Polish Academy of Sciences.
References GUZEV, W. S., KULICHEVSKAYA, 1. S., and ZVYAGINTSEV, D. G.: Skaninguyushchaya elektronnaya mikroskopiya pri izucheniy wzaimodieystviya mikroorganizmov s rastieniyami. In: Mikro· organizmy kak komponenty biogeocenoza (E. N. MISHUSTIN, ed.) Moscow 1984, 92-107. JONES, P. C. T., and MOLLISON, J. N.: A technique for the quantitative estimation of soil microorganisms. J. Gen. Microbiol. 2 (1948), 54-69. KACZMAREK, W., KASZUBIAK, R., and GUZEK, R.: Comparison of changes in the number of microorganisms in the soil by the plate and microscopic procedures. Pol. J. Soil Sci. 6 (1973), 133 to 139. KASZUBIAK, R., DURSKA, G., KACZMAREK, W., and FILODA, G.: Effect of slurry on microorganisms and chemical properties of soil. Zbl. Mikrobiol. 138 (1983), 501-509. - KACZMAREK, W., and Plj)DZIWILK, Z.: Comparison of different methods fo estimating thes productivity of microorganisms in soil. Ekol. Pol. 25 (1977), 289-296. - and MUSZYNSKA, M.: Bacteria of meadow sward and the effect of slurry dressing on their community. Zentralbl. Mikrobiol. 142 (1987), 13-21. - and RATAJCZAK, R.: Wplyw bromoxynilu na drobnoustroje w ryzosferze j~czmienia. PTPN, Prace Komisji Nauk Roln. i Komisji Nouk Lesnych 35 (1973), 185-189.
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H. KASZUBIAK and M. MUSZYNSKA, Bacterial Number and Biomass in a Meadow
MUSZYNSKA, M., and KASZUBIAK, H.: The influence of slurry on the bacterial biomass in incubated soil samples. Zbl. Mikrobiol. 140 (1985), 277-282. STRUGGER, S.: A fluorescent microscope examination of bacteria in soil. Can. J. Res., Sec. C, 26 (1948),188-193. TEsARovA, M.: Bakterienbiomasse in verschieden bewirtschafteten Wiesen-Okosystemen. Zbl. Mikrobiol. 138 (1983), 179-186. WALLACE, R. H., and LOCHHEAD, A. G.: Qualitative studies of soil microorganisms. IX. Amino acid requirements of rhizosphere bacteria. Can. J. Res., Sec. C 28 (1950), 1-6. WEST, P. M., and LOCHHEAD, A. G.: Quantitative studies of soil microorganisms. IV. The rhizosphere in relation to the nutritive requirements of soil bacteria. Can. J. Res. 18 (1940),129-136. ZVYAGINTSEV, D. G., and ROGACHEVSKIY, L. M.: Plotnost (udielniy vies) kletok mikroorganizmov. Mikrobiologiya 42 (1973), 892-898. Authors' address: Prof. Dr. HENRYK KASZUBIAK and Dr. MARIA MUSZYNSKA, Department of Agricultural Microbiology, Academy of Agriculture, W olynska 35. 60-637 Poznan. Poland.