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Fluctuations in physiological groups of bacteria in the horizons of a beech forest soil
Soil Biol. Biochem. Vol. 15, NO. 1, pp. 45550, 1983 Printed in Great Britain. All rights reserved
0038-0717/83/010045-06$03.00/O Copyright Q 1983 Pergamon Press Ltd
FLUCTUATIONS IN PHYSIOLOGICAL GROUPS OF BACTERIA IN THE HORIZONS OF A BEECH FOREST SOIL TIIU KAURI University
of Lund, Department of Microbial Ecology, Ecology Helgonavagen 5, S-223 62 Lund, Sweden
Building,
(Accepted 20 June 1982)
Summary-The population densities of physiological groups of bacteria were studied in a beech forest soil in order to assess the role of bacteria as decomposer organisms in the ecosystem. A direct multipoint method was used to estimate the numbers of bacteria in different physiological groups. Large numbers of bacteria were found decomposing xylan and pectin. The numbers of each physiological group differed considerably between horizons. Fluctuations in numbers within physiological groups were greater in the upper horizons. No consistent patterns of fluctuations caused by seasonal changes could be observed. A defoliation of the forest by Dasychira pudibundu larvae in the middle of the experimental period resulted in a significant increase in numbers of chitin decomposers and a small decrease of starch hydrolyzers
INTRODUCTION
pectin, starch, cellulose and chitin was studied in various horizons of a beech forest soil. The seasonal variation in the total population and in bacterial (endo-) spores in this soil has been described previously (Kauri, 1982). As the experimental conditions were similar in both studies the present report on horizonal and seasonal variation of the physiological groups of bacteria can be directly compared with the study on total numbers of bacteria.
The decomposition of organic matter on forest floors is important for the re-circulation of nutrients in the forest ecosystem. Plants contain a high proportion of starch, which is the most common reserve nutrient in plants, cellulose, hemicelluloses, such as xylan and pectin, and lignin. Chitin is the most important structural component of animals found in soil, mainly in arthropod exoskeletons, but it is also present in the cell walls of many fungi. Large quantities of these polymers reach the soil every year, primarily as plant residues, but they are also added to the soil pools as dead eukaryotic and prokaryotic cells. In order to explain the degradation of these compounds in natural habitats, more information is required about the distribution and density of the decomposer organisms in soil. Fungi, which are often considered the most important decomposer group in soil (Satchel], 1971), have attracted much attention (Anderson and Domsch, 1975), but information about bacterial decomposers has been sparse (Gyllenberg and Eklund, 1974; Swift et al., 1979). Recently, some evidence has been obtained that ascribes to bacteria a greater role than known before in the initial decomposition of organic materials (Thayer and Murray, 1977; Sreenath et al., 1978). Furthermore, Deschamps et al. (1980) concluded that bacteria are active degraders of wood, which suggests a modification of our opinion about bacterial biodegrading abilities. One of the problems in studies of bacteria as decomposer organisms has been the lack of suitable experimental methods. For this purpose, a most probable number method was developed for use with a multipoint inoculator on agar plates (Kauri, 1980). In the present investigation, the ability of aerobic bacteria, including actinomycetes, to degrade xylan,
MATERIALS AND METHODS
The fluctuations of bacterial populations were followed in the four horizons of a beech forest soil during a 3-yr period (1972-l 975). In the middle of this period (summer, 1973) the forest was invaded by a large population of Dasychira pudibundu L. larvae, which caused earlier defoliation of the trees (Kauri, 1982). Consequently, the entire forest was already defoliated in September, whereas usually the peak litter-fall does not occur until late in October. During this period numerous dead pale tussock moth larvae were found under the trees on the forest floor. Site and sampling
The site is located in Kongalund (55?59’N, 13”10’E), South Sweden. The mature beech (Fugus syhxztica L.) grew on an old forest area with acid brown forest soil. The abundant understory vegetation in spring was followed by sparse summer vegetation. Mean annual temperature is 67°C and mean annual precipitation is about 800 mm for this area. The description of the research area, climatical factors and some characteristics of the soil profile, such as soil moisture content, organic matter, and pH of the different horizons, was given in some detail previously 45
TIIU KAURI
46
(Kauri, 1982). The soil analyses were conducted on the same soil samples as in the present study. The acid soil had a pH (H20) of about 4, with small variations between horizons. The soil samples were collected from the soil profile and divided into the following horizons: hO, A,,/A,, A, and (B). Estirnution
of the physiologiml
groups
qf bacteria
Soil suspensions were made with 20g fresh soil or litter in Winogradsky’s standard salt solution (Pochon, 1954) as described previously (Kauri, 1980). Various dilutions were made for dilferent horizons, depending on the predicted bacterial numbers in each horizon. Five serial soil dilutions and five replicates from each dilution were inoculated by a multipoint inoculator from a plastic tray (master tray) with 25 compartments onto agar plates containing specific substrates utilizable by different physiological groups (Kauri, 1980)The same master tray was used for inoculation of all of the physiological groups in the same horizon. The plates were incubated at 20°C in an inverted position in closed compartments to reduce dehydration. The incubation time allowed for the degradation of starch was 14 days, of pectin 3 weeks, of xylan 8 weeks, and of chitin and cellulose 10 weeks. The media used in the agar plates for the determination of the physiological groups of bacteria were specified by Kauri (1980). After incubation, the positive sites (degradation zones. maximum 25 on each plate) were counted on the five replicate plates used for each of the physiological groups. For the estimation of the numbers in each physiological group, a most probable number method (MPN), described by Kauri (1980), was used. Special MPN tables were prepared for different dilution series, where numbers of positive sites on a plate corresponded to MPN values. The numbers of bacteria were then calculated using the MPN values, inoculation volume, and dry weight of the soil sample.
RESULTS
Due to the attack by pale tussock moth larvae in the summer of 19’73, the reported observations became distributed over an initial pre-attack period, until September 1973, and a perturbed, post-attack period, after September 1973. Significant differences were found in numbers of bacteria between horizons for ail physiological groups (Student’s t-test). The fluctuations of the physiological groups are shown as numbers of bacteria in Fig. I and as percent of the total aerobic bacteria in Fig. 2. Although the initial unperturbed period proved to be unduly short, as a consequence of the defoliation it reveals the relative importance and the fluctuations of the physiological capacities of the bacterial populations in a general way. The fluctuations of cellufcrse-decomposers were not very high during the different seasons (Fig. I). The numbers diminished with depth and their proportion of the total aerobic bacteria (Kauri, 1982) were
similar between the horizons (Fig. 2). In terms of absolute numbers, the cellulose-decomposers were one of the smallest groups in every horizon. Among starch hydrolyzers, the highest differences in numbers were found in the A, horizon. The numbers decreased in all horizons after defoliation of the beech trees by pale tussock moth larvae. Between seasons, the Ao,/A, horizon showed the highest fluctuations during the first part of the investigation period, before defoliation, with low numbers during the summer and peaks in autumn and spring. The A, horizon had a small peak in autumn, and the (B) horizon had a peak in both spring and autumn. The relative numbers of starch hydrolyzers, expressed as percent of total aerobic bacteria, were much lower in the A,, than in the other horizons (Fig. 2). The A, horizon showed the highest fluctuation in relative numbers. In the (B) horizon, the percentage of starch hydrolyzers was high during the first year and then decreased to a low level after the defoliation. The A,,/A, horizon showed an intermediate state. The numbers of chitin degraders in the A,,, horizon did not fluctuate much until the post-attack period, in the spring of 1974, when the numbers increased markedly and remained high. In the A,,/A, horizon the numbers of chitin hydrolyzers increased immediately after the defohation and remained high throughout the rest of the sampling period. The A, horizon also showed little fluctuation in chitin hydrolyzers until the increase after the defoliation. The (B) horizon has the smallest fluctuations and the lowest numbers of chitin hydrolyzers. in spite of the increase during the post-attack period. When the numbers of chitin utilizers were expressed as percent of the total aerobic bacteria, there was little dilference between the horizons and the fluctuations were small in the pre-attack period. After the defoliation, however, there was a marked increase in the relative numbers of chitin utilizers, w-hich rose from ca. 20% to 507; in all horizons. The numbers of pectin and _~ylun hydrolyzers showed similarities in their fluctuations, probably because they represent similar ecological groups of bacteria that degrade hemicelluloses. Both groups fluctuated markedly and generally had the same pattern. The upper horizons had the highest numbers of these hydrolyzers in autumn, and the deeper horizons had smaller fluctuations and highest numbers late in the year. The (B) horizon had the lowest numbers and the smallest fluctuations. The percentages of pectin and xylan degraders were much higher than those of the other physiological groups (Fig. 2) and they reflected, more or less, the fluctuation in the total aerobic bacterial population (Kauri, 1982). When the physiological groups of bacteria were compared to the total aerobic bacterial populations, by means of linear regression analyses, significant correlations were obtained in all horizons for pectin and xylan hydrolyzers (Table 1). Thus. it is probable that these groups are of particular importance in the fluctuation in bacterial numbers found in the present investigation. Fluctuations in chitin hydrolyzers were not significantly correlated with fluctuations in total
Physiological
groups
of bacteria
in forest
soil
1o-7 236
I
200-
150-
l
-
c
pectin xylan
,FYIYJJA~OND~JCYIYJJASOND~,J9~;AYJJ1SOND~
1972
1975
1973
Date
Fig. I. Fluctuations in numbers of bacteria in various physiological groups (starch, pectin, xylan, chitin and cellulose) in different soil horizons during the investigation period. The SEM was less than I lyO.
TIIU KAURI
48
aerobic bacteria, and the correlation coefficient decreased with depth (Table 1). Significant correlations were obtained for the cellulolytic bacteria in both the A,,/A, and A,, horizons, whereas a very low significance was found in the deeper horizons. Starch hydrolyzers showed a low correlation rate with the aerobic bacteria, and for the A, horizon no significant correlation was found. The linear regression analyses included all sampling periods entailing a probable influence from the defoliation perturbation.
DISCUSSION
The relatively high numbers of bacteria in various physiological groups obtained in this study compared to other investigations (Goodfellow, 1968; Hissett and Gray, 1974) could be due to the direct inoculation with soil solutions. The method excluded selective steps in the isolation procedure of the decomposer organisms that could diminish their numbers or decomposing capacities and allowed association with other organisms for the benefit of decomposition. Evidence of
u1 /U
80
60
40
c
-
A,,
t-.
A,,/A,
l
----- A,
-
(6)
-“(: J
\\
xylan
1972
1974
1973
1975
Date
Fig. 2. Percent
of different
physiological groups of the total aerobic bacterial horizons during the investigation period.
population
in various
Physiological groups Table
I, Correlation
Am *,,,:A, A, (B\
of bacteria
in forest
between physiological groups of bacteria in soil horizons
0.76* 0.54* 0.46 0.75**
0.87*** o.E!3*** 0.61* 0.79***
0.96*** 0.79** 0.76* 0.89***
soil
49
and total aerobic
0.47 0.34 -0.24 0.06
bacteria
0.80** 0.73** 0.53* 0.55*
***p < 0.001. **p < 0.01. *P < 0.05. this effect has recently been provided for pectin (Pickaver, 1977) and xylan hydrolyzers (Sreenath et al., 1978). As some bacteria have the ability to decompose several compounds, they can be included in more than one group. Thus, the sum of bacteria in all physiological groups will combine to a larger total number than that represented by the plate counts. In nature cellulose degradation is a relatively slow process as compared to degradation of other compounds (xylan, pectin, starch) investigated here that are readily hydrolyzed by many different bacteria. Chitin, like cellulose, occurs in the matrix with other structural compounds and offers more resistance to degradation. Therefore, it is not surprising that bacteria capable of degrading cellulose and chitin comprise only a small part of the total aerobic bacterial population. The constant presence of cellulose and chitin in forest soils probably provides a continuous supply of the essential requirements for these more and therefore only small stable populations, fluctuations in their numbers occur. As degradation is influenced by substrate availability, the notable increase in the chitin pool during the post-attack period led to a significant increase in chitinolytic bacteria. The increase was similar in all horizons, but bacterial numbers were at a higher level in the upper horizons. The increase in the chitin pool was probably the result of the larvae that were lying in masses around the trees or of the possible increase in fungal activity because of the defoliation or both. Also, in a laboratory experiment Veldkamp (1955) showed as a result of chitin addition to soil an increase in chitin decomposing bacteria. The influence of substrate availability on population densities is also exemplified by the increase of numbers of bacteria in specific physiological groups late in the year after litter fall (organic input) and probably by the decrease of bacterial numbers in physiological groups down the horizons with decreasing and not so readily decomposable (recalcitrant) organic matter content. The numbers of bacteria capable of hydrolyzing starch showed only limited seasonal fluctuations, but their percentage of the total bacterial population increased with depth, suggesting that most of the substrate available for starch hydrolyzers probably comes from the roots. Defoliation appeared to have caused a reduction of the relative numbers of starch hydrolyzers during the post-attack period. During the year of defoliation the trees must economize on their starch reserves, as they most likely did not have time before defoliation to collect the starch from the leaves.
Webb (1980) studied starch content as a measure of reserve energy of conifers defoliated by Douglas-fir tussock moth. He found in consequence a reduced starch content in all vegetative parts of the trees. As defoliation also could cause reduced photosynthetic capacity (Kulman, 1971) the starch content available for microbial degradation would then be smaller. This agrees with the results obtained here, where decreasing populations of starch hydrolyzers were observed in all horizons after the defoliation. In a Danish beech forest, Holm and Jensen (1972) found that actinomycetes comprised I SO;;,of the bacterial isolates in surface soil and that the percentage increased with depth. In the present investigation the actinomycetes were included among soil bacteria, and were frequently found on the substrate plates utilizing starch, pectin, xylan, chitin and cellulose. The actinomycetes seem to be important components of the decomposing bacterial flora. Their frequent occurrence might be due to the direct inoculation of soil samples (Kauri, 1980). This is supported by Goodfellow and Cross (1974), who suggested that the low numbers and few species of actinomycetes reported from litter studies may be a result of inappropriate isolation techniques. The method used here excluded the isolation step that may have made it possible for actinomycetes to exhibit their degrading abilities. In contrast to former studies (Waksman, 1922; Jensen, 1930), lately actinomycetes have been found to be widespread in acid soils (Khan and Williams, 1975; Goodfellow and Dawson, 1978), thus corroborating the results of the present study. Khan and Williams (1975) found the majority to be streptomycetes, able to degrade hemicellulose and other polysaccharides, and therefore actinomycetes were assumed to be important in decomposition processes in acid soils. In conclusion, the quantities, distribution and fluctuation of bacteria of different physiological capabilities in the soil horizons were studied to explore the role of bacteria as decomposer organisms of organic material in natural habitats. Large populations of bacteria were found decomposing especially xylan and pectin in soil. The different physiological groups were more exposed to fluctuations in the surface horizons. As part of nutrient availability, the influence on the physiological groups was illustrated by the invasion of Dasychirn pudibunda larvae. Acknowledgements--I thank Professor G. Stotzky, Dr B. Nordbring-Hertz and Dr B. Siiderstri_jm for their constructive criticism. The multipoint-inoculator was constructed by Dr T. Rosswall.
50
Tnu KAIJRI REFERENCES
Anderson J. P. E. and Domsch K. H. (1975) Measurement of bacterial and fungal contributions to respiration of selected agricultural and forest soils. Canadian Journal of Microbiology 21, 3 14-322. Deschamps A. M., Mahoudeau G., Leulliette L. and Lebeault J.-M. (1980) Isolation and identitication of bark decaying and utilizing bacteria of various origins. Revue d’Ecologir et de Biologic du Sol 17, 577-581. Goodfellow M. (1968) Properties and composition of the bacterial flora of a pine forest soil. Journal of‘Soil Science 19, 154-167. Goodfellow M. and Cross T. (1974) Actinomycetes. In Biology of Plant Lifter Decomposition (C. H. Dickinson and G. J. F. Pugh, Eds), pp. 2699302. Academic Press, London. Goodfellow M. and Dawson D. (1978) Qualitative and quantitative studies of bacteria colonizing Picea sitchensis litter. Soil Biology & Biochemistry 10, 3033307. Gyllenberg H. G. and Eklund E. (I 974) Bacteria. In Biology of Plum Litter Decomposirion (C. H. Dickinson and G. J. F. Pugh, Eds), pp. 245-268. Academic Press, London. Hissett R. and Gray T. R. G.(1974) Bacterial populations of litter and soil in p deciduous woodland. I. Quantitative studies. Rewe d’Ecologie et de Biologic du Sol 4,495-508. Holm E. and Jensen V. (I 972) Aerobic chemoorganotrophic bacteria of a Danish beech forest. Oikos 23, 248-260. Jensen H. L. (1930) Actinomycetes in Danish soils. Soil Science 30, 59-77. Kauri T. (1980) Rapid multipoint method for quantification of various physiological groups of bacteria in siol. Soil Biology & Bioche&/ry Ii, 1255130. Kauri T. (1982) Seasonal fluctuations in numbers of aerobic bacteria and’their spores in four horizons of a beech forest soil. Soil Biology & Biochemistry 14, 185-190.
Khan M. R. and Williams S. T. (1975) Studies on the ecology of actinomycetes in soil-VIII. Distribution and characteristics of acidophilic actinomycetes. Soil Biology & Biochemistry 7, 345-348. Kulman H. M. (1971) Effects ofinsect defoliation on growth and mortality of trees. Annual Reriebt, of’ Entomology 16, 289. Pickaver A. H. (I 977) Diagnostic agar plate techniques for testing pectinase-producing bacteria can give false negative results. FEMS Microbiology Letlers 2, 105~107. Pochon J. (1954) Munwl Technique d’Anal~w Microhiologique du Sol. Masson et Cie. Paris, Satchel1 J. E. (1971) Feasibility study of an energy budget for Meathop Wood. In Productivity of Forest Ecosystems, No, 4. Proceedings of the Brussels Symposium (P. Duvigneaud. Ed.). Paris. UNESCO. Sreenath H. K.. Joseph R. and Murthy V. S. (1978) Studies on xylan hydrolases from different strains of S/ren/omvces and their mutual influences in the breakdown of xylan. Folia Microbiologica 23, 299-303. Swift M. J., Heal 0. W. and Anderson J. M. (1979) Decomposition in Terrestrial Ecosystems. Studi<~s in Ecology. Vol. 5. Blackwell. Oxford. Thayer D. W. and Murray J. 0. (1977) Physiological, biochemical and morphological characteristics of mesquite wood-digesting bacteria. Journal of Cenerol Microbiology 101,71-77. Waksman S. A. (1922) The influence of soil reaction upon the growth of actinomycetes causing potato scab. Soil Science 14, 61-79. Webb W. L. (1980) Starch content of conifers defoliated by the Douglas-fir tussock moth. Cunudion Journal o/‘Foresr Research 10, 5355541. Veldkamp H. (1955) A study of the aerobic decomposition of chitin by microorganisms. Medelingrn run de Lundbouuhogeschool te Wageningen 55, I21- 174.
0038-0717/83/010045-06$03.00/O Copyright Q 1983 Pergamon Press Ltd
FLUCTUATIONS IN PHYSIOLOGICAL GROUPS OF BACTERIA IN THE HORIZONS OF A BEECH FOREST SOIL TIIU KAURI University
of Lund, Department of Microbial Ecology, Ecology Helgonavagen 5, S-223 62 Lund, Sweden
Building,
(Accepted 20 June 1982)
Summary-The population densities of physiological groups of bacteria were studied in a beech forest soil in order to assess the role of bacteria as decomposer organisms in the ecosystem. A direct multipoint method was used to estimate the numbers of bacteria in different physiological groups. Large numbers of bacteria were found decomposing xylan and pectin. The numbers of each physiological group differed considerably between horizons. Fluctuations in numbers within physiological groups were greater in the upper horizons. No consistent patterns of fluctuations caused by seasonal changes could be observed. A defoliation of the forest by Dasychira pudibundu larvae in the middle of the experimental period resulted in a significant increase in numbers of chitin decomposers and a small decrease of starch hydrolyzers
INTRODUCTION
pectin, starch, cellulose and chitin was studied in various horizons of a beech forest soil. The seasonal variation in the total population and in bacterial (endo-) spores in this soil has been described previously (Kauri, 1982). As the experimental conditions were similar in both studies the present report on horizonal and seasonal variation of the physiological groups of bacteria can be directly compared with the study on total numbers of bacteria.
The decomposition of organic matter on forest floors is important for the re-circulation of nutrients in the forest ecosystem. Plants contain a high proportion of starch, which is the most common reserve nutrient in plants, cellulose, hemicelluloses, such as xylan and pectin, and lignin. Chitin is the most important structural component of animals found in soil, mainly in arthropod exoskeletons, but it is also present in the cell walls of many fungi. Large quantities of these polymers reach the soil every year, primarily as plant residues, but they are also added to the soil pools as dead eukaryotic and prokaryotic cells. In order to explain the degradation of these compounds in natural habitats, more information is required about the distribution and density of the decomposer organisms in soil. Fungi, which are often considered the most important decomposer group in soil (Satchel], 1971), have attracted much attention (Anderson and Domsch, 1975), but information about bacterial decomposers has been sparse (Gyllenberg and Eklund, 1974; Swift et al., 1979). Recently, some evidence has been obtained that ascribes to bacteria a greater role than known before in the initial decomposition of organic materials (Thayer and Murray, 1977; Sreenath et al., 1978). Furthermore, Deschamps et al. (1980) concluded that bacteria are active degraders of wood, which suggests a modification of our opinion about bacterial biodegrading abilities. One of the problems in studies of bacteria as decomposer organisms has been the lack of suitable experimental methods. For this purpose, a most probable number method was developed for use with a multipoint inoculator on agar plates (Kauri, 1980). In the present investigation, the ability of aerobic bacteria, including actinomycetes, to degrade xylan,
MATERIALS AND METHODS
The fluctuations of bacterial populations were followed in the four horizons of a beech forest soil during a 3-yr period (1972-l 975). In the middle of this period (summer, 1973) the forest was invaded by a large population of Dasychira pudibundu L. larvae, which caused earlier defoliation of the trees (Kauri, 1982). Consequently, the entire forest was already defoliated in September, whereas usually the peak litter-fall does not occur until late in October. During this period numerous dead pale tussock moth larvae were found under the trees on the forest floor. Site and sampling
The site is located in Kongalund (55?59’N, 13”10’E), South Sweden. The mature beech (Fugus syhxztica L.) grew on an old forest area with acid brown forest soil. The abundant understory vegetation in spring was followed by sparse summer vegetation. Mean annual temperature is 67°C and mean annual precipitation is about 800 mm for this area. The description of the research area, climatical factors and some characteristics of the soil profile, such as soil moisture content, organic matter, and pH of the different horizons, was given in some detail previously 45
TIIU KAURI
46
(Kauri, 1982). The soil analyses were conducted on the same soil samples as in the present study. The acid soil had a pH (H20) of about 4, with small variations between horizons. The soil samples were collected from the soil profile and divided into the following horizons: hO, A,,/A,, A, and (B). Estirnution
of the physiologiml
groups
qf bacteria
Soil suspensions were made with 20g fresh soil or litter in Winogradsky’s standard salt solution (Pochon, 1954) as described previously (Kauri, 1980). Various dilutions were made for dilferent horizons, depending on the predicted bacterial numbers in each horizon. Five serial soil dilutions and five replicates from each dilution were inoculated by a multipoint inoculator from a plastic tray (master tray) with 25 compartments onto agar plates containing specific substrates utilizable by different physiological groups (Kauri, 1980)The same master tray was used for inoculation of all of the physiological groups in the same horizon. The plates were incubated at 20°C in an inverted position in closed compartments to reduce dehydration. The incubation time allowed for the degradation of starch was 14 days, of pectin 3 weeks, of xylan 8 weeks, and of chitin and cellulose 10 weeks. The media used in the agar plates for the determination of the physiological groups of bacteria were specified by Kauri (1980). After incubation, the positive sites (degradation zones. maximum 25 on each plate) were counted on the five replicate plates used for each of the physiological groups. For the estimation of the numbers in each physiological group, a most probable number method (MPN), described by Kauri (1980), was used. Special MPN tables were prepared for different dilution series, where numbers of positive sites on a plate corresponded to MPN values. The numbers of bacteria were then calculated using the MPN values, inoculation volume, and dry weight of the soil sample.
RESULTS
Due to the attack by pale tussock moth larvae in the summer of 19’73, the reported observations became distributed over an initial pre-attack period, until September 1973, and a perturbed, post-attack period, after September 1973. Significant differences were found in numbers of bacteria between horizons for ail physiological groups (Student’s t-test). The fluctuations of the physiological groups are shown as numbers of bacteria in Fig. I and as percent of the total aerobic bacteria in Fig. 2. Although the initial unperturbed period proved to be unduly short, as a consequence of the defoliation it reveals the relative importance and the fluctuations of the physiological capacities of the bacterial populations in a general way. The fluctuations of cellufcrse-decomposers were not very high during the different seasons (Fig. I). The numbers diminished with depth and their proportion of the total aerobic bacteria (Kauri, 1982) were
similar between the horizons (Fig. 2). In terms of absolute numbers, the cellulose-decomposers were one of the smallest groups in every horizon. Among starch hydrolyzers, the highest differences in numbers were found in the A, horizon. The numbers decreased in all horizons after defoliation of the beech trees by pale tussock moth larvae. Between seasons, the Ao,/A, horizon showed the highest fluctuations during the first part of the investigation period, before defoliation, with low numbers during the summer and peaks in autumn and spring. The A, horizon had a small peak in autumn, and the (B) horizon had a peak in both spring and autumn. The relative numbers of starch hydrolyzers, expressed as percent of total aerobic bacteria, were much lower in the A,, than in the other horizons (Fig. 2). The A, horizon showed the highest fluctuation in relative numbers. In the (B) horizon, the percentage of starch hydrolyzers was high during the first year and then decreased to a low level after the defoliation. The A,,/A, horizon showed an intermediate state. The numbers of chitin degraders in the A,,, horizon did not fluctuate much until the post-attack period, in the spring of 1974, when the numbers increased markedly and remained high. In the A,,/A, horizon the numbers of chitin hydrolyzers increased immediately after the defohation and remained high throughout the rest of the sampling period. The A, horizon also showed little fluctuation in chitin hydrolyzers until the increase after the defoliation. The (B) horizon has the smallest fluctuations and the lowest numbers of chitin hydrolyzers. in spite of the increase during the post-attack period. When the numbers of chitin utilizers were expressed as percent of the total aerobic bacteria, there was little dilference between the horizons and the fluctuations were small in the pre-attack period. After the defoliation, however, there was a marked increase in the relative numbers of chitin utilizers, w-hich rose from ca. 20% to 507; in all horizons. The numbers of pectin and _~ylun hydrolyzers showed similarities in their fluctuations, probably because they represent similar ecological groups of bacteria that degrade hemicelluloses. Both groups fluctuated markedly and generally had the same pattern. The upper horizons had the highest numbers of these hydrolyzers in autumn, and the deeper horizons had smaller fluctuations and highest numbers late in the year. The (B) horizon had the lowest numbers and the smallest fluctuations. The percentages of pectin and xylan degraders were much higher than those of the other physiological groups (Fig. 2) and they reflected, more or less, the fluctuation in the total aerobic bacterial population (Kauri, 1982). When the physiological groups of bacteria were compared to the total aerobic bacterial populations, by means of linear regression analyses, significant correlations were obtained in all horizons for pectin and xylan hydrolyzers (Table 1). Thus. it is probable that these groups are of particular importance in the fluctuation in bacterial numbers found in the present investigation. Fluctuations in chitin hydrolyzers were not significantly correlated with fluctuations in total
Physiological
groups
of bacteria
in forest
soil
1o-7 236
I
200-
150-
l
-
c
pectin xylan
,FYIYJJA~OND~JCYIYJJASOND~,J9~;AYJJ1SOND~
1972
1975
1973
Date
Fig. I. Fluctuations in numbers of bacteria in various physiological groups (starch, pectin, xylan, chitin and cellulose) in different soil horizons during the investigation period. The SEM was less than I lyO.
TIIU KAURI
48
aerobic bacteria, and the correlation coefficient decreased with depth (Table 1). Significant correlations were obtained for the cellulolytic bacteria in both the A,,/A, and A,, horizons, whereas a very low significance was found in the deeper horizons. Starch hydrolyzers showed a low correlation rate with the aerobic bacteria, and for the A, horizon no significant correlation was found. The linear regression analyses included all sampling periods entailing a probable influence from the defoliation perturbation.
DISCUSSION
The relatively high numbers of bacteria in various physiological groups obtained in this study compared to other investigations (Goodfellow, 1968; Hissett and Gray, 1974) could be due to the direct inoculation with soil solutions. The method excluded selective steps in the isolation procedure of the decomposer organisms that could diminish their numbers or decomposing capacities and allowed association with other organisms for the benefit of decomposition. Evidence of
u1 /U
80
60
40
c
-
A,,
t-.
A,,/A,
l
----- A,
-
(6)
-“(: J
\\
xylan
1972
1974
1973
1975
Date
Fig. 2. Percent
of different
physiological groups of the total aerobic bacterial horizons during the investigation period.
population
in various
Physiological groups Table
I, Correlation
Am *,,,:A, A, (B\
of bacteria
in forest
between physiological groups of bacteria in soil horizons
0.76* 0.54* 0.46 0.75**
0.87*** o.E!3*** 0.61* 0.79***
0.96*** 0.79** 0.76* 0.89***
soil
49
and total aerobic
0.47 0.34 -0.24 0.06
bacteria
0.80** 0.73** 0.53* 0.55*
***p < 0.001. **p < 0.01. *P < 0.05. this effect has recently been provided for pectin (Pickaver, 1977) and xylan hydrolyzers (Sreenath et al., 1978). As some bacteria have the ability to decompose several compounds, they can be included in more than one group. Thus, the sum of bacteria in all physiological groups will combine to a larger total number than that represented by the plate counts. In nature cellulose degradation is a relatively slow process as compared to degradation of other compounds (xylan, pectin, starch) investigated here that are readily hydrolyzed by many different bacteria. Chitin, like cellulose, occurs in the matrix with other structural compounds and offers more resistance to degradation. Therefore, it is not surprising that bacteria capable of degrading cellulose and chitin comprise only a small part of the total aerobic bacterial population. The constant presence of cellulose and chitin in forest soils probably provides a continuous supply of the essential requirements for these more and therefore only small stable populations, fluctuations in their numbers occur. As degradation is influenced by substrate availability, the notable increase in the chitin pool during the post-attack period led to a significant increase in chitinolytic bacteria. The increase was similar in all horizons, but bacterial numbers were at a higher level in the upper horizons. The increase in the chitin pool was probably the result of the larvae that were lying in masses around the trees or of the possible increase in fungal activity because of the defoliation or both. Also, in a laboratory experiment Veldkamp (1955) showed as a result of chitin addition to soil an increase in chitin decomposing bacteria. The influence of substrate availability on population densities is also exemplified by the increase of numbers of bacteria in specific physiological groups late in the year after litter fall (organic input) and probably by the decrease of bacterial numbers in physiological groups down the horizons with decreasing and not so readily decomposable (recalcitrant) organic matter content. The numbers of bacteria capable of hydrolyzing starch showed only limited seasonal fluctuations, but their percentage of the total bacterial population increased with depth, suggesting that most of the substrate available for starch hydrolyzers probably comes from the roots. Defoliation appeared to have caused a reduction of the relative numbers of starch hydrolyzers during the post-attack period. During the year of defoliation the trees must economize on their starch reserves, as they most likely did not have time before defoliation to collect the starch from the leaves.
Webb (1980) studied starch content as a measure of reserve energy of conifers defoliated by Douglas-fir tussock moth. He found in consequence a reduced starch content in all vegetative parts of the trees. As defoliation also could cause reduced photosynthetic capacity (Kulman, 1971) the starch content available for microbial degradation would then be smaller. This agrees with the results obtained here, where decreasing populations of starch hydrolyzers were observed in all horizons after the defoliation. In a Danish beech forest, Holm and Jensen (1972) found that actinomycetes comprised I SO;;,of the bacterial isolates in surface soil and that the percentage increased with depth. In the present investigation the actinomycetes were included among soil bacteria, and were frequently found on the substrate plates utilizing starch, pectin, xylan, chitin and cellulose. The actinomycetes seem to be important components of the decomposing bacterial flora. Their frequent occurrence might be due to the direct inoculation of soil samples (Kauri, 1980). This is supported by Goodfellow and Cross (1974), who suggested that the low numbers and few species of actinomycetes reported from litter studies may be a result of inappropriate isolation techniques. The method used here excluded the isolation step that may have made it possible for actinomycetes to exhibit their degrading abilities. In contrast to former studies (Waksman, 1922; Jensen, 1930), lately actinomycetes have been found to be widespread in acid soils (Khan and Williams, 1975; Goodfellow and Dawson, 1978), thus corroborating the results of the present study. Khan and Williams (1975) found the majority to be streptomycetes, able to degrade hemicellulose and other polysaccharides, and therefore actinomycetes were assumed to be important in decomposition processes in acid soils. In conclusion, the quantities, distribution and fluctuation of bacteria of different physiological capabilities in the soil horizons were studied to explore the role of bacteria as decomposer organisms of organic material in natural habitats. Large populations of bacteria were found decomposing especially xylan and pectin in soil. The different physiological groups were more exposed to fluctuations in the surface horizons. As part of nutrient availability, the influence on the physiological groups was illustrated by the invasion of Dasychirn pudibunda larvae. Acknowledgements--I thank Professor G. Stotzky, Dr B. Nordbring-Hertz and Dr B. Siiderstri_jm for their constructive criticism. The multipoint-inoculator was constructed by Dr T. Rosswall.
50
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