Influence of plants and cropping on microbiological characteristics of some new Caledonian ultramafic soils.
Five New Caledonian ultramafic soils were compared for their bacterial and fungal population densities and for their microbial activity estimated by fluorescein diacetate (FDA) hydrolysis. The low microbial numbers and activities were related to the organic matter content and to metallic toxicity. Actinomycetes were found to be dominanant among bacterial populations. The effect of soil colonisation by plants on the microflora was studied and appeared to be very favourable. The rhizosphere effect of 2 plant species (Grevillea gillivrayi and Costularia comosa) was estimated. The influence of cropping on the development of microflora in one soil was also investigated and a qualitative study of the fungal populations and their variation in relation to the cropping was reported. The fungal flora was dominated by Moniliaceae, and Tuberculariaceae and Dematiaceae were absent in natural ultramafic soils. Cropping enhanced the diversity of these fungal populations.
Additional keywords: biological activity, fungi, microflora, metals.
In New Caledonia, outcrops of ultramafic rocks occupy nearly one third of the total surface of the island. This surface is colonised by a special serpentine vegetation with a very high level of endemism (Brooks 1987). Nickel mining is the main economic activity. Over the past decades, mining has led to degradation of large areas of soil and vegetation and to pollution of rivers (Jaffre and Rigault 1991). The revegetation of these areas is now widely recognised to be essential but cost-effective methods of achieving this need to be developed. Indeed, the topsoil has been destroyed in many places, and the underlying layer is not only very poor but also toxic to plants because of its high metal content.
Some studies on this subject suggest methods for revegetation (Williamson et al. 1982; Bradshaw 1984; Seaker and Sopper 1988; Jaffre et al. 1993; Jasper 1994) but these need to be adapted and improved. An alternative method involves the cropping of some ultramafic soils after improvement. Thus, scientific studies of the New Caledonian ultramafic soils, their microflora, and their activity are necessary to gain an understanding of how plant growth can be stimulated and, when possible, to propose ways of improving soil fertility. Basic knowledge of the microflora of New Caledonian ultramafic soils is lacking, although these microorganisms are very interesting because of their adaptive features.
This paper presents a preliminary overview of the microflora of some New Caledonian ultramafic soils and discusses the influence of plants and cropping on this microflora.
Materials and methods
Six ultramafic soils were studied. Four were topsoils from scrublands with serpentine vegetation: a hypermagnesian brown soil (HBS) from Plum, a gravelly ferralitic oxidic soil (GFOS) from Ouenarou, a cropped ferralitic oxidic soil (CrFOS) from Ouenarou, and a colluvial ferralitic oxidic soil (CFOS) from `Plaine des Lacs'. Two were subsoils from the `Tontouta' mine in an exploited area without topsoil: a lateritic subsoil (lat.) and a saprolitic subsoil (sapro.).
The main physico-chemical characteristics of these soils are given in Table 1.
[TABULAR DATA 1 NOT REPRODUCIBLE IN ASCII]
Soil sites and sampling
Soil samples were collected from the top 20 cm soil layer. The 4 topsoils were generally taken between plants where there were few fine roots.
In order to study the influence of soil colonisation by plants, samples were collected on a transect extending from a cluster of plants (including Alphitonia neocaledonica, Costularia comosa, and Pteridium esculatum) to a bare patch, on a flat and homogeneous site. Five points were marked every 1.5 m and 4 samples were taken at each point. In order to estimate the rhizosphere effect of 2 plant species (Grevillea gillivrayi and Costularia comosa), samples of fine roots with adhering soil were collected under 4 plants of each species. A non-rhizospheric soil, without roots, was also sampled at the same depth.
To study the influence of cropping and fertilisation on the evolution of the soil microflora, samples were taken from a field cropped with corn(*). The ferralitic oxidic soil had been cropped for about 2 years before sampling. It had been amended with 1.5 t/ha of pounded calcareous crust (with 44% CaO) to reduce Ca deficiency, and fertilized with 500 kg/ha of urea (in 3 applications) and 142 kg/ha of potassium sulfate.
Four samples were collected from 3 trial plots under corn shoots: a plot fertilized with phosphorus pentoxide ([P.sub.2][O.sub.5]) at 4 t/ha, another with the same compound at 10 t/ha, and a plot fertilised with [P.sub.2][O.sub.5] (4 t/ha) and enriched with composted leaves (20 t/ha).
Other soil samples were taken from a ploughed, non-planted soil and from a natural, non-ploughed soil adjacent to the experimentation field. Samples were also collected between 2 corn crops, 3 weeks after a sorghum intermediate crop had been ploughed in.
Soil microbial population count
Each soil sample was sieved through 2-mm mesh and analysed immediately. For the rhizosphere study, the roots were shaken slowly by hand to recover the adhering soil which was considered as the rhizosphere.
Determination of the microbial densities was performed by the dilution-plate count technique. Only mesophilic, organotrophic, and aerobic microflora were studied (anaerobic microflora is probably poor because these soils are porous and frequently dry). Soil (10 g) was suspended in water (90 mL) and shaken vigorously for 15 min. Appropriate series of 10-fold dilutions were prepared and aliquots of 1 mL were placed in Petri dishes before the medium was added.
For bacteria and actinomycetes, a peptone-yeast extract -- dextrose -- agar medium was used. The pH was adjusted to 7. Filter-sterilised actidione (50 mg/L) was added after sterilisation by autoclave. Actinomycetes were distinguished from the other bacteria by the dry aspect of their colonies. When necessary, a microscopic observation was made. For fungi, the composition of the medium was malt extract (10 g), dextrose (5 g), agar (20 g), and mineralised water (1000 mL); 100 mg/L of filter-sterilised streptomycin was added after sterilisation by autoclave. Colonies were counted after 4-6 days of incubation at 25 [degrees] C.
Qualitative study of the microflora
Bacteria and actinomycetes were isolated and some were purified. The percentage of Gram negative and Gram positive bacteria was estimated and the predominant genera of actinomycetes were determined according to the Bergey's manual of determinative bacteriology (Williams et al. 1989).
The different genera of fungi were identified after microscopic observation according to the manuals by Barnett and Hunter (1986), Barron (1977), and Ellis (1993).
Soil microbial activity as measured by FDA hydrolysis
The enzymatic method is frequently used for comparative studies of total soil activity (Schnurer and Rosswall 1982; Zelles et al. 1987). The FDA (fluorescein diacetate) test was performed as described by Bailly-Marion (1993): soil (1 g) was added to phosphate buffer (10 mL), pH 7.6 ([KH.sub.2][PO.sub.4] 0.1 N, [Na.sub.2][HPO.sub.4] 0.1 N) and homogenised by strong agitation (vortex) for 1 min before being incubated for 20 min at 23 [degrees] C. To the soil suspension was added 1 mL FDA (200 mg, acetone 60 mL, distilled water up to 100 mL). The tubes were incubated again for 40 min at 23 [degrees] C in the dark. The reaction was stopped by adding 1 mL of [HgCl.sub.2] solution (0.4 mg/mL in distilled water). The soil was removed from the solution by centrifugation at 2500G for 10 min and filtered through Whatman paper GF/F. The absorbance was measured with a spectrophotometer at 490 nm. The amount of fluorescein in the filtrate was determined from a standard curve (490 nm) and FDA hydrolysis activity was expressed as nmol of fluorescein/h.g soil (dry weight).
Statistical analyses were performed by computer with the program statview 4.02 (Abacus Concepts Inc. 1993).
The microbial populations of the 5 ultramafic soils were small compared with more classical soils, and varied greatly (Fig. 1). The largest population was found in the HBS which had > 1.5 x [10.sup.6] CFU/g of soil bacteria and > 3 x [10.sup.4] CFU/g of soil fungi. In comparison, the GFOS contained less than one-fifth the amount of bacteria and nearly the same quantity of fungi. The aerobic microflora of the CFOS was represented mainly by actinomycetes (> [10.sup.4] CFU/g). These latter microorganisms were dominant with more than 75% of the total counted bacteria in the 3 topsoils. The lateritic and saprolitic subsoils had < [10.sup.4] CFU/g of soil microorganisms and the actinomycetes represented < 30% of the total counted bacteria. The fungal population levels were > 600 CFU/g of soil (saprolitic) and 100 CFU/g of soil (lateritic).
[FIGURE 1 GRAPH OMITTED]
The total microbial activity (FDA) of the 5 ultramafic soils was low compared with an agricultural soil (318.9 nmol/g soil) (Table 2). The HBS showed the highest activity (> 69 nmol fluorescein/g dry soil). The 2 ferralitic oxidic soils had similar values (about two-thirds that of HBS). The activity levels of the 2 mine subsoils were the lowest with only 15.1 nmol fluorescein/h.g dry soil for the saprolitic subsoil. The influence of plants and litter on FDA activity was strongly positive.
Table 2. FDA (fluorescein diacetate) hydrolytic activity of the soils
For comparison, the FDA activity of a New Caledonian agricultural soil [see Amir and Pineau (1997) for physical and chemical characteristics) was tested and the value obtained was 318.9 nmol/g dry soil. Values followed by the same letter are not significantly different at P = 0.05 (variance analysis for all values taken together). HBS, hypermagnesian brown soil; GFOS, gravelly ferralitic oxidic soil; CFOS, colluvial ferralitic oxidic soil
Soil FDA hydrolitic activity (nmol fluorescein/h.g dry soil) Bare soil Bare soil Soil under litter without roots with roots with roots HBS 68.9 f 98.6 cd 240.3 a GFOS 46.7 g 84-9 e 123.4 bc CFOS 49.8 g 94.4 de 159.8 b Lateritic subsoil 26.9 h n.d. n.d. (mine) Saprolitic subsoil 15.1 i n.d. n.d. (mine)
n.d., not determined (there were no plants in these areas).
The FDA activity of the 5 soils was positively correlated to the microbial densities. The coefficient of correlation between this activity and log(total bacteria) was r = 0.857 (significant at P = 0.001); the same coefficient between FDA activity and log(fungal flora) was r = 0.742 (P = 0.002).
The colonisation of GFOS by plants supported higher microbial populations (Fig. 2). The number of bacteria and fungi decreased gradually on a transect from a cluster of plants to a bare site. For example, the bacterial density of soil under plants (5.7 x [10.sup.5] CFU/g) was > 7 times higher than that for bare soil. The number of actinomycetes was not affected by the absence of vegetation, but the other bacteria were very rare in the soil without roots. The level of fungal populations in the bare soil (4.0 x [10.sup.3] CFU/g of soil) was about 20 times lower than that under plant litter.
[FIGURE 2 ILLUSTRATION OMITTED]
The rhizosphere of the 2 plants (Grevillea gillivrayi and Costularia comosa) appeared to have a strong positive influence on microbial density (Fig. 3). The R/S ratios (rhizosphere/non rhizospheric soil) were higher for fungi than for the other 2 groups. The high percentage of actinomycetes in the control soil (85%) decreased strongly in the rhizosphere (13-30%). The rhizosphere effect of Costularia comosa seemed to be somewhat greater than that of Grevillea gillivrayi.
[FIGURE 3 GRAPH OMITTED]
Gram negative bacteria were predominant in the ultramafic soils, especially in the 2 ferralitic oxidic soils which contained only 28% Gram positive bacteria. In HBS, Gram positive bacteria represented 43%. The actinomycetes found in these soils were dominated by the 2 common genera Streptomyces and Streptoverticillium, which represented more than 70% of the actinomycetal flora.
Table 3 shows the fungal genera identified and their relative importance in the 5 soils studied. Moniliaceae were the best represented family. The genera Aspergillus and Penicillium generally accounted for over 50% of the microflora. Trichoderma, Paecilomyces, Scopulariopsis, and Cephalosporium were also present in addition to small numbers of Phycomycetes, especially Pythium sp., Mucor sp., and Mortierella sp.
[TABULAR DATA 3 NOT REPRODUCIBLE IN ASCII]
Several differences between the 5 soils were noted. The HBS did not contain Penicillium spp. In the absence of vegetation, GFOS was dominated by the genus Aspergillus but, under litter, the number of genera increased and Penicillium spp. became more common than Aspergillus spp. Penicillium spp. were also dominant in the rhizosphere of Grevillea gillivrayi which also seemed to have a positive effect on Pythium sp. and some other species.
Despite their very low organic matter content (Table 1), the lateritic and saprolitic subsoils contained a number of fungal genera, especially the latter, from which 10 different genera were observed.
The influence of cropping, organic enrichment, and [P.sub.2 O.sub.5] fertilisation of a ferralitic oxidic soil on the density of soil microflora is shown in Table 4. On the whole, the natural non-ploughed soil contained fewer microorganisms than the ploughed and non-planted soil (39.1 X [10.sup.4] and 85.0 x 10.sup.4] bacteria/g respectively). However, whilst the number of actinomycetes was approximatively the same in the 2 soils (30.6 x [10.sup.4] and 34.0 x [10.sup.4] CFU/g), the percentage of actinomyeetes in the natural soil was very high (about 80%). In the soil samples collected under the corn shoots, the bacterial population was 3 times higher than in the non-ploughed soil but the actinomycetes were not significantly stimulated by the crop. The fungal density (20 x [10.sup.3] CFU/g in the non-ploughed soils) showed little increase under the corn crop. The 2 [P.sub.2 O.sub.5] applications (4 and 10 t/ha) had approximately the same moderately positive influence on the microflora in the cropped soil, and the addition of compost (20 t/ha) before cropping did not improve the number of microorganisms.
[TABULAR DATA 4 NOT REPRODUCIBLE IN ASCII]
On the other hand, the organic input provided by a sorghum crop, ploughed in between 2 corn crops, had a considerable influence on microbial density: the bacterial population rose to 398 x [10.sup.4]/g and the number of actinomycetes increased by a factor of 4. The mycoflora was also stimulated, but less than the bacteria. After this enrichment, an application of 10 t/ha Of [P.sub.2 O.sub.5] increased the microbial density more markedly than the smaller dose (4 t/ha).
Cropping also had a notable effect on the qualitative features of the mycopora (Table 5). Two genera of Dematiaceae and Tuberculariaceae (Cladosporium and Cylindrocarpon) which were not represented in the natural non-ploughed soil, were found in very small numbers on the cropped soils. A few genera of these 2 families (Drechslera, Humicola, unidentified Dematiaceae, and Fusarium) were isolated in soils where sorghum had been ploughed in, between 2 corn crops. The genus Aspergillus was not from any of the cropped soil plots, whereas it was relatively abundant (10-50%) in the natural soil.
[TABULAR DATA 5 NOT REPRODUCIBLE IN ASCII]
It is important to point out some shortcomings associated with the dilution-plate method we have used. It is well known that the total number of soil microflora obtained with this technique is always underestimated (Paul and Clark 1988). In addition to the problem of microbial aggregation, different groups of bacteria and fungi do not grow in laboratory media (Dommergues and Mangenot 1970). In our study, only aerobic organotrophic microorganisms were counted. Despite these drawbacks, comparisons between different samples remain valid. In addition, this method shows some important advantages, especially the possibility of studying the qualitative aspects of the microflora. When completed with microbial activity, this method can useful results.
Despite their differences, the 5 soils studied all have in common a low level of organic matter (ranging from traces to 2%), a high metal content, and lack of structure. These characteristics help to explain the low microbial densities and activities, since organic matter content and microbial biomass are known to be correlated (Chaussod et al. 1986). Furthermore, Amir and Pineau (1998) found evidence of toxic effects of metals, especially nickel, on microbial development in these soils. On the whole, HBS, which has more organic carbon than the other soils, contained the largest number of microorganisms and showed the highest FDA activity. The lateritic and saprolitic subsoils, both very poor in carbon, contained the lowest number of bacteria and actinomycetes and showed the lowest activities. It is therefore interesting to note that HBS showed the highest level of extractable Ni and Mn, which could explain the limitation of microbial growth and FDA activity in this soil despite its markedly higher organic matter content compared with the other soils. There was a high positive correlation between FDA hydrolytic activity and microbial population densities, especially the bacterial flora (r = 0.857; P [is less than] 0.001). However, GFOS, which had over 10 times more microorganisms than the colluvial soil, had approximatly the same FDA activity. This latter soil is richer in organic matter but contains more extractable toxic metals. The combined effect of these 2 factors may explain the differences between microbial densities and FDA activities of the 2 soils. The total numbers of microorganisms in the 3 topsoils (HBS, GFOS, and CFOS) are similar to those reported by Williams et al. (1977) in a mining soil containing high levels of Pb and Zn (about 2 x [10.sup.5] CFU/g soil). It is difficult to compare the microbial activities obtained here with those found by other authors since there are no reports where the same method was used for similar soils, and the values of activities are only relative.
One of the peculiar characteristics of the 3 topsoils is the high percentage of actinomycetes, which represent 70-90% of the total microflora. The same dominance of actinomycetes was reported by Amir et al. (1985) for Saharian soils and could be related to the ability of these microorganisms to resist extreme conditions and to decompose complex substances like lignin and humus (Dommergues and Mangenot 1970). We found actinomycetes to be more tolerant to metals than bacteria (Amir and Pineau 1998). However, other authors reported them to be less resistant to metal toxicity, than bacteria and fungi (Jordan and Lechevalier 1975; Williams et al. 1977; Hiroki 1992).
In the soils colonised by plants and in the rhizosphere of the 2 plant species studied, the proportion of actinomycetes dropped to a low level, possibly as a result of the presence of easily degradable energetic compounds in root exudates and cell decomposition for which bacteria are more competitive (Alexander 1977).
The qualitative aspect of the mycoflora of the New Caledonian ultramafic soils is also interesting, although it must be remembered that the dilution-plate method is somewhat unsatisfactory in that it favours the highly sporulating fungi. Despite this drawback, some characteristics are sufficiently clear to be emphasised, the most important being the predominance of Moniliaceae which represent 90% or more of the total fungal population. Another feature is the non-isolation of some families which are usually very common in soils, especially the Tuberculariaceae such as Fusarium spp. and the Dematiaceae such as Alternaria spp. No samples of New Caledonian ultramafic soils analysed to date contained fungi of these 2 families. This might be related to metal toxicity, since 2 strains of Fusarium oxysporum and Curvularia lunata have proved to be more sensitive to nickel than other fungi (Amir and Pineau 1998).
It is difficult to establish similarities between our findings and those of other authors because the features of the soils studied differ so widely. However, it could be useful to mention that Williams et al. (1977) emphasised the dominance of another fungal group, the mucorales (especially the genus Mortierelia), in a mining soil with high levels of Pb and Zn. An abundance of mucorales has also been reported by Wong et al. (1978) in soil polluted with iron-ore tailings.
Natural plant growth in ultramafic soils appeared to stimulate the microflora and its activity more than in a classic soil, since in ultramafic soils, roots and litter greatly increase the amount of organic matter, which is initially very low. In all cases, FDA activity of the soil from under plants was significantly more important. The presence of litter also strongly stimulates this activity. Organic matter improves the soil structure and reduces the toxic effect of metallic ions through adsorption (Hattori and Hattori 1976). The rhizosphere effect of the 2 plants studied is also very important because of the almost total lack of high energy compounds in the ferralitic oxidic soil.
The influence of cropping was beneficial to microbial development in the ferralitic oxidic soil. Bacteria were stimulated more than actinomycetes and fungi. This stimulation could be caused by a number of different factors such as soil watering, soil fertilisation, and, above all, the increase of the organic matter content after several crops (Table 4 shows the difference between the ploughed non planted soil and the cropped soil). The ploughing in of sorghum, 3 weeks before our count was made, resulted in a proliferation of all microbial groups, especially bacteria. The diversity of the fungal population was also enhanced as Tuberculariaceae (Fusarium) spp. and Dematiaceae (Drechslera sp., Humicola sp.), which had not been found previously, appeared in the soil enriched with sorghum straw. Few publications have dealt with the influence of cropping or organic inputs on microbial populations of extreme soils. Seaker and Sopper (1988) showed that enrichment of a coal mine spoil by municipal sludge resulted in a large increase in bacterial, fungal, and actinomycetal populations and their activity.
The results recorded in this study should be regarded as preliminary data in the microbiology of New Caledonian ultramafic soils and further more detailed research in this field is required. Some original characteristics of the microflora of these soils have been pointed out, especially the abundance of actinomycetes, the lack of certain fungal groups, and the great influence of plants and organic matter on the evolution of these immature soils. The importance of organic matter in ultramafic soils has to be taken into account in revegetation after mining. Another microbiological aspect, which has not been discussed here, is the very important role of arbuscular mycorrhizal fungi which is now under study. These fungi appear to be crucial to many serpentine plant species in New Caledonia (Amir et al. 1997), showing similarities to soils of Australian mining regions (Jasper 1994). Actinomycetes of ultramafic soils were found to be indigenous and to produce several antibiotics (H. Amir, D. Saintpierre, R. Pineau, unpublished data); current research is directed towards these substances.
This research was supported mainly by the South Province of New Caledonia (Grant no. 239 PVF/DDR). The authors thank Dr Bonzon (ORSTOM, New Caledonia) and Dr Pelletier (SLN, New Caledonia) for their cooperation and advice, and for the physicochemical analyses of the soils.
(*) The field experimentation was conducted by soil scientists of ORSTOM (Institut Francis de Recherche Scientifique pour le Development en Cooperation), Noumea and CREA (Centre de Recherche et d'Experimentations Agronomiques), Bourail.
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|Author:||Amir, H.; Pineau, R.|
|Publication:||Australian Journal of Soil Research|
|Date:||May 1, 1998|
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