Agriculture, Ecosystems and Environment 112 (2006) 1–12
www.elsevier.com/locate/agee
Soil algae composition under different agro-ecosystems
in North-Eastern Italy
Santina Zancan a, Renata Trevisan b,*, Maurizio G. Paoletti b
a
Dipartimento di Biotecnologie Agrarie Università di Padova, Viale dell’Università 16, 35020 Legnaro (Pd), Italy
b
Dipartimento di Biologia Università di Padova, via U. Bassi 58/b, 35121 Padova, Italy
Received 1 December 2003; received in revised form 1 April 2005; accepted 22 June 2005
Available online 4 October 2005
Abstract
Soil algae can perform important services for agro-ecosystems and functions as a bioindicator for soil quality. Communities of topsoil
algae were studied (species composition and counts) in four different agro-ecosystems in relation to different land uses, including tillage. The
chosen agro-ecosystems were an intensively-cultivated corn (Zea mays L.) field, a vineyard, a pasture and a field abandoned for 12 years. In
all, 92 algal species were identified in the different ecosystems, which were sampled twice (March and May). In 2001 the main algal species
were Cyanophyceae, Chlamydophyceae, and Chlorophyceae. The greatest species richness of algal species was recorded in the pasture,
followed by the abandoned field, the corn field and the vineyard. Using a dilution method on solid media to count the algae enabled the
distinction and separate quantification of cyanophytes, diatoms and greenish algae (cholorophytes (CH) + xanthophytes (X) + eustigmatophytes (E)). This last group of greenish algae dominated at all four sites, together with cyanophytes in the pasture and the abandoned field,
whereas cyanophytes were almost absent in the vineyard. The highest mean abundance of algal cells in the 0–2 cm soil layer was found in the
abandoned field and in the pasture, while it was lower in the corn field and in the vineyard. The undisturbed sites (abandoned field and, to some
extent, pasture) were also characterised by a greater diversity of algae. Disturbance (tillage and use of pesticides) seemed to have a strong
effect on both the composition and the density of the algal communities. Cyanophytes (Calothrix sp., Cylindrospermum sp., Pseudoanabaena
sp., Scytonema sp. and Thricormus sp.) seemed to be particularly sensitive to disturbance. Heavy metals residues in the soil, especially Cu
(linked to fungicide sprays, used particularly in vineyards), may be a factor affecting the abundance of cyanobacteria. It would be reasonable
to conclude that the structure of soil algal communities is affected more by soil use rather than by physico-chemical soil parameters.
Cyanobacteria showed the most evident response in the different agro-ecosystems, and therefore seem to be the most suitable group to
consider as a soil bioindicator of land use.
# 2005 Elsevier B.V. All rights reserved.
Keywords: Agro-ecosystems; Bioindicators; Cyanobacteria; Rural environments; Soil-algae
1. Introduction
Algae (both eukaryotics and cyanobacteria) occupy a
variety of terrestrial habitats, including soils, rocks and
caves; they inhabit permanent snow and ice fields, and can
also be found on living animals and plants (Hoffmann,
1989).
Soil habitats are the most important non-aqueous
ecosystems for algae (Zenova et al., 1995). The activities
* Corresponding author. Tel.: +39 049 8276293; fax: +39 049 8276230.
E-mail address: [email protected] (R. Trevisan).
0167-8809/$ – see front matter # 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.agee.2005.06.018
of algae contribute to soil formation, to the stability of
mature soils (Metting, 1981), and to the energy and matter
fluxes in ecosystems (Kuzyakhmetov, 1998a). Another
important aspect of soil algae is nitrogen fixation. Algae
contribute to the nitrogen content of the soil through the
process of biological nitrogen fixation (Goyal, 1997).
Green and blue-green populations in upper topsoil are
large and diverse, and they can perform valuable services for
the soil ecosystems (Metting, 1981; Starks et al., 1981) and
for agriculture too (Ruble and Davis, 1988). One of the
major benefits of algal functions in terrestrial habitats is the
product of their photoautotrophic nutrition, the generation of
2
S. Zancan et al. / Agriculture, Ecosystems and Environment 112 (2006) 1–12
organic matter from inorganic substances (Alexander,
1977). They also serve as a food source for bacteria and
invertebrates, and biologically active compounds produced
by algae can affect other components of soil communities,
including plants (Metting, 1981; Zenova et al., 1995).
Estimates of soil algae biomass in temperate lands are
usually expressed as the number of cells per gram of dry soil,
or as the number of cells per square metre of soil. In the
former case, the algal biomass varies from 0 to 108 cells g1
soil (d.w.) (Ruble and Davis, 1988; Sukala and Davis, 1994;
Wöhler et al., 1998; Lukešová, 2001), while in the latter it
ranges from 0 to 1011 cells m2 (Shimmel and Darley, 1985;
Lukešová, 1993; Lukešová and Hoffmann, 1996). Soil algal
biomass can sometimes be expressed as kilograms of
biomass per soil area. Boul et al. (1972) indicated a mean
value of 10 kg ha1.
Algae represent the first community to colonise bare soil,
including volcanic deposits and unfarmed soils from various
origins, thereby enabling the subsequent establishment of
higher plant communities (Starks et al., 1981). The role of
algae as pioneer organisms is particularly important in bare
soils liable to erosion (Booth, 1941). Soil algae, especially
cyanobacteria, are known to aggregate soil particles by
producing extracellular polysaccharides (Lynch and Bragg,
1985) and forming water-stable aggregates that reduce the
impact of wind erosion (Johansen, 1993).
The nature of algal flora in different localities is the result
of a complex influence of the local type of vegetation, soil
properties and climatic conditions (Metting, 1981; Starks
et al., 1981; Lukešová, 1993), but it often also depends on
the input of airborne algal diasporas (Brown et al., 1964).
The interaction of soil biota, e.g. micro-organisms, roots,
animals and plants is affected, sometimes significantly, by
agricultural practices and human activities. Soil labour,
pesticide residues, chemical fertilisers and agronomic
practices affect plants, animal life and soil community
structure (Paoletti et al., 1988).
Despite numerous studies on soil algae (Metting, 1981;
Starks and Shubert, 1981; Starks et al., 1981; Johansen,
1993; Lukešová, 1993; Sukala and Davis, 1994; Lukešová
and Hoffmann, 1996; Tsujimura et al., 2000; Lukešová,
2001; Neustupa, 2001), it is still difficult to draw general
conclusions on the diversity of the flora and their influence
on ecosystem functions. Algal biomass estimates in soil
populations differ greatly because there are no standard
methods as regards counting or the use of enrichment media
for instance (Hoffmann, 1989; Tsujimura et al., 2000).
It is assumed that changes in land management influence
biota as revealed by certain transient or permanent signs
detectable in the system of biological communities under
consideration (Paoletti and Pimentel, 1992). Studies
concerning the use of bioindicators have suggested
biodiversity as a basic indicator of landscape quality and
a fundamental tool for assessing the impact and success of
remediation processes. Limits to its practicability (using
biodiversity as an indicator) are due to our limited
knowledge of the micro-organisms populating the ecosystems (Paoletti, 1999).
The aim of this study was to identify and quantify the soil
algae at four sites characterised by the same soil type but
differing in their land use history. Specific objectives were to
(1) test the effect of land use and tillage intensity on soil
algal densities, community structure and composition; (2)
assess the potential for using algae as bioindicator for soil
quality under different soil management strategies; and (3)
investigate the influence of soil temperature and humidity on
soil algal densities and composition.
2. Materials and methods
2.1. Study area description
To assess the reaction of soil algal communities to
agricultural practices, four sites subjected to different
degrees of tillage were compared. All four sites are located
at the ‘‘Lucio Toniolo’’ Experimental Agricultural Station of
the Faculty of Agriculture, University of Padova, NorthEastern Italy (458120 N, 118580 E) at 6 m a.s.l. (Fig. 1). This
area is characterised by a mean annual temperature of
12.5 8C, a mean temperature in the warmest month of
22.1 8C and mean temperature in the coldest month of
1.3 8C. The annual temperature range indicates a sub
continental regime. The mean annual precipitation is
838 mm, concentrated mostly between April and November
(Scotton et al., 2000; Ziliotto et al., 1996).
All the sites are on the same soil type, classified as fluvicalcaric-cambisol.
The physical characteristics of the four sites are described
below:
1. Abandoned field—in the area, where the University
buildings now stand, there was a grassland up until 1990.
Fig. 1. Location of sampling sites in north-east Italy. (a) Abandoned field;
(p) pasture; (c) corn field; (v) vineyard; (1) University buildings; (2) ‘‘Lucio
Toniolo’’ Experimental Agricultural Station; (3) residential area.
S. Zancan et al. / Agriculture, Ecosystems and Environment 112 (2006) 1–12
Excavation for the buildings’ foundations gave rise to
several large heaps of soil that were abandoned for about
6 years. In 1997, the soil was spread to level ground and
has since been left undisturbed. The original soil in this
area was turned several times and then abandoned, so the
horizons became mixed together. At the time of
sampling, there was a ruderal and discontinuous
vegetation comprising only volunteer plants, and many
areas with no vegetation cover.
2. Pasture—up until 1982, this site was farmed intensively
(corn (Zea mays L.), sugar beet (Beta vulgaris L.), wheat
(Triticum aestivum L.), then it was seeded with a mixture
of graminaceous plants. From 1982 to 1999, the grasses
were undisturbed, except for mowing three to four times a
year. In 2000, the site was used as grazing land for cattle.
At the time of sampling, the grass was about 15 cm high
in March and about 120 cm high in May.
Given the lack of tillage and most other kinds of
human activity in recent years, the abandoned field and
pasture can be considered as scarcely-disturbed sites.
3. Vineyard—this site was planted 30 years ago. The grass
is mowed regularly in spring and summer, and exposed to
conventional pesticide inputs. In 2000, it was treated five
times with Mancozeb fungicide (2 kg ha1) and, after
flowering, with cupric antiperonospora products
(2.5 kg ha1). Sulphur was added to all the treatments
(2.0 kg ha1). In 2001, between the first and second
sampling, the vineyard was treated twice (10th–18th
May) with Mancozeb (2 kg ha1) and copper oxychloride (2.5 kg ha1). At the time of sampling, plant growth
was poor, about 5 cm long in March and 15–20 cm in
May. There was plant litter on the topsoil.
4. Corn field—field farmed intensively with wheat in 1997,
soybean (Glycine max (L.) Merr.) in 1998 and corn in
both 1999 and 2000. The conventional, high-input
method applied to this site included a twice-yearly
mineral fertilisation, one before seeding (N–P–K) and
one at the time of seeding (N), and a winter ploughing
(depth 35–40 cm). During the winter before the
sampling, the plot was fertilised with manure
(50,000 kg ha1) and, about 20 days before the first
sampling was collected, the plot was ploughed and
3
uprooted. Between the two sampling collections (on 25th
April), the plot was seeded (corn) and herbicides applied
(5 l ha1 of Metolaclor (322 g/l) and Terbutilazina
(161 g/l)).
The vineyard and corn field were considered exposed to
intensive tillage.
All sites were assumed to have identical climatic
conditions. Details of the characteristics of the sites are
given in Table 1. Meteorological data for the sites were
obtained from the weather station at ‘‘Lucio Toniolo’’
Experimental Agricultural Station about 1 km away.
2.2. Soil sampling
Samples were collected twice, on 7th March and 22nd
May 2001, during the vegetation period, the interval
between the two dates being long enough to feature clearly
different meteorological conditions, but short enough to
minimize the tillage operations performed in the meantime.
Twenty soil subsamples per sample were taken randomly
from different places at each site and combined together for
subsequent analysis. Subsamples were taken from the soil
surface layer (0–2 cm) using a blade sterilised with ethanol
after each use. The samples were placed in sterile bags and
carried to the laboratory, where they were placed in a dark
room kept at 4 8C. At the two sampling times, soil
temperature were measured 10, 5 and 1 cm below the soil
surface and also on the soil surface using a digital
thermometer.
2.3. Laboratory analysis
Laboratory analysis on the samples began within 24 h of
the collection. The soil was sieved to remove pebbles,
fragments of vegetation (including roots) and, if necessary,
larger invertebrates. A 10 g portion of each sample was
mixed with water at a ratio of 1:1 and soil pH was
determined after 1 h (Hunt et al., 1979) using a pH-meter
with a glass combination electrode. Chemical soil parameters were analysed in the samples collected in May. The
available phosphorus (Pi) was extracted in a sodium
Table 1
Temperature and moisture of soils from abandoned field, pasture, vineyard and corn field (mean and standard deviation)
Soil depth (cm)
Abandoned field
mean (S.D.)
Pasture
mean (S.D.)
Vineyard
mean (S.D.)
Corn field
mean (S.D.)
Temperature (8C)
March
May
March
May
1
1
5
5
14.6
25.6
13.1
24.3
11.0 (0.75)
18.8 (0.47)
9.7 (0.48)
18.3 (1.63)
12.1
20.8
10.4
19.9
12.1 (0.96)
27.9 (2.12)
8.9 (0.48)
22.9 (1.67)
Moisture (g kg1)
March
May
0–2
0–2
217 (15)
174 (21)
315 (25)
276 (14)
441 (25)
346 (24)
(0.7)
(1.0)
(0.5)
(0.7)
(0.99)
(1.96)
(0.58)
(0.92)
184 (17)
193 (11)
4
S. Zancan et al. / Agriculture, Ecosystems and Environment 112 (2006) 1–12
bicarbonate solution and measured spectrophotometrically
(Watanabe and Olsen, 1965). Total nitrogen (Ntot) was
measured by the Kjeldahl method (Bremner and Mulvaney,
1982). Organic carbon (Corg) was oxidized with a sulphuric
acid and potassium bichromate mixture; excess potassium
bichromate was titrated with a Mohr salt (Nelson and
Sommers, 1982). The K, Ca, Na, Cu, Cd, and Zn contents of
the soil were extracted with 1 M ammonium acetate and
measured by flame absorption spectrophotometry (Thomas,
1982). Soil texture (mass of sand-silt-clay-sized particles)
was determined using a hydrometer. Freshly sieved samples
were weighed onto dried aluminium trays, heated at 105 8C
for 48 h, and reweighed to determine moisture loss. This
data was used to convert all algal counts from number per
gram of wet weight to number per gram of dry weight of soil
(Table 2).
2.4. Soil algae
Culture media included Bold’s Basal Medium (BBM),
BG-11 with or without (BG-118) combined nitrogen and
Water Soil Medium (WSM) (Stein, 1969). These media were
used in liquid or solidified form with 1.5% agar. One
millilitre of 0.11 N solution of Na2SiO39H2O was added to
each litre of BBM to provide silicon for diatoms (Sukala and
Davis, 1994). Media were sterilised for 15 min in an
autoclave and then transferred into either sterile test tubes or
sterile plastic Petri dishes.
Dilution on solid media was chosen for algal counts:
10 g of soil were transferred to 90 ml of sterile water and
homogenised. Then serial 4-fold dilutions of homogenate
in liquid BBM were prepared. Each Petri dish containing
solidified BBM was inoculated with 1 ml of diluted soil
suspension; 4 replication of each dilution were used
(Lukešová, 1993). Incubation proceeded for 3 weeks in the
environmental chamber equipped with fluorescent tubes
(Philips ‘‘TL’’ D 840) provided light at 14 h/10 h light-
dark cycle. The chamber temperature was kept at 23–
25 8C. The number of algae was estimated by counting the
algal colonies that developed on agar plates of the most
suitable dilution (20–400 colonies/plate), taking 1 cell = 1
colony. Colonies of Chlorophyta (CH), Xanthophyceae
(X) and Eustigmatophyceae (E) (hereinafter CH + X + E)
were counted together because they are difficult to
distinguish (Lukešová, 1993). Bacillariophyceae (D) and
Cyanophyceae (CY) were counted separately. The number
of algae was estimated per 1 g dry weight (d.w.) of soil
(Fig. 2).
Two procedures were used to identify the algae. First, the
colonies grown on BBM plates for algae quantification
purposes and colonies grown on other plates with BG11,
BG118 and WSM were examined microscopically. Most of
the algae recorded in the plates were not identifiable by direct
observation, so it was necessary to isolate the strains into
unialgal cultures and to study their life cycles to ensure a
correct species identification. Isolates were obtained by
transferring a small amount of the cells from a colony to a
sterile test tube filled with liquid BBM and leaving them to
grow for 2–4 weeks in the incubation chamber before any
identification was attempted. Second, the ‘‘growth slides’’
method was used (Lund, 1945). Soil samples were placed in
sterile Petri dishes, covered with sterile cover slips, moistened
with sterile water and incubated in the incubation chamber.
The cover slips were removed and actively-growing algae
adhering to them were examined under the microscope.
Diatom frustules were cleaned by soaking in hydrogen
peroxide (H2O2) overnight. They were then rinsed and
centrifuged repeatedly. All nondiatom algae were identified
by direct microscopic examination of wet mounts. Iodinepotassium iodide solution (IKI) was used to determine the
presence of starch, pyrenoids and flagella. All organisms
were studied and photographed using an Olympus BH 2
light microscope. Standard taxonomic references were
consulted for most identifications.
Table 2
Chemical properties of soils from abandoned field, pasture, vineyard and corn field (mean and standard deviation)—soil depth 0–2 cm
pH
C org (g kg1)
N tot (g kg1)
C/N
Pi (mg kg1)
K (mg g1)
Ca (mg g1)
Na (mg g1)
Sand (g kg1)
Silt (g kg1)
Clay (g kg1)
Cu (mg g1)
Cd (mg g1)
Zn (mg g1)
Abandoned field
mean (S.D.)
Pasture
mean (S.D.)
Vineyard
mean (S.D.)
Corn field
mean (S.D.)
7.73 (0.23)
4.4 (0.2)
1.4 (0.06)
3.2 (0.11)
29 (0.73)
79 (5.06)
120 (6.24)
65 (4.55)
460 (31)
300 (22)
240 (13)
15.1 (0.98)
0.1 (0.01)
25.6 (1.48)
7.61 (0.31)
33.9 (3.2)
2.4 (0.1)
13.8 (0.94)
69 (6.0)
183 (10.1)
135 (9.27)
68 (4.69)
440 (38)
320 (16)
240 (22)
21.6 (1.15)
0.3 (0.02)
52.6 (4.26)
7.40 (0.38)
37.7 (2.9)
3.1 (0.2)
13.3 (0.69)
74 (8.14)
793 (54.6)
173 (8.04)
72 (6.12)
340 (14)
350 (24)
310 (30)
52.6 (4.52)
0.5 (0.04)
13.2 (0.96)
7.96 (0.35)
10.0 (0.7)
1.7 (0.1)
5.9 (0.15)
68 (2.58)
234 (10.8)
125 (11.5)
71 (5.04)
420 (23)
280 (19)
300 (25)
37.8 (2.42)
0.3 (0.02)
43.1 (3.93)
S. Zancan et al. / Agriculture, Ecosystems and Environment 112 (2006) 1–12
5
Fig. 2. Photographs of soil algae found (bar = 10 mm): (a) Klebsormidium flaccidum (Kützing) Silva, Mattox and Blackwell (very common soil algal species
found at all studied sites); (b) Cylindrospermum sp. (cyanophycean algae found only in pasture); (c) Eustigmatos magnus (J.B. Petersen) Hibberd (common soil
species found at all studied sites except the corn field); (d) Hantzschia amphioxys (Ehren.) Grunow in Cleve and Grunow (widespread soil diatom found at all
studied sites); (e) Nitzschia palea (Kützing) W. Smith (common soil diatom found at all studied sites except the corn field); (f) Xanthonema montanum (Vischer)
Silva (widespread soil species found at all studied sites).
2.5. Statistical analysis
Data reported concerning algae abundance and physicochemical parameters are the mean standard deviation
(S.D.) of four independent samples.
Data analyses were performed using SPSS for
Windows Version 8 (SPSS UK Ltd., Woking, UK) (Foster,
1998). The unweighted pair-group method using Jaccard’s
coefficient of similarity and based on a matrix representing species presence/absence data was performed to
compare the algal communities from the four sites at the
two sampling times. A cluster analysis based on the
presence/absence matrix enabled a dendrogram to be
obtained. A correlation analysis was performed to select
uncorrelated variables fit for variance analysis. If the
Pearson’s rho coefficient significance level was >0.05 the
variable couple could be considered uncorrelated. A first
analysis of the principal components was based on
uncorrelated soil characteristics. A second was conducted
to compare the four sites on the basis of algal community
characteristics, i.e. total algal density, CH + X + E, CY
and D density, number of taxa identified belonging to
different algal classes.
3. Results
3.1. Site conditions
The two sets of samples collected in March and May were
characterized by different meteorological conditions (total
rainfall during the 20 days before sampling: 228 mm in
March and 506 mm in May, mean daytime maximum air
temperature during the 20 days before sampling: 11.4 8C in
March and 25.3 8C in May, mean daytime minimum air
temperature during the 20 days before sampling: 0.7 8C in
6
S. Zancan et al. / Agriculture, Ecosystems and Environment 112 (2006) 1–12
Table 3
Composition (species and number) of algal communities of abandoned field, pasture, vineyard and corn field at the two sampling times
Cyanophyceae
Anabaena sp.1
Calothrix sp.
Cylindrospermum sp.
Nostoc sp.1
Nostoc sp.2
Scytonema sp.
Trichormus sp.
Heterocystous sp.1
Heterocystous sp.2
Ilyonema sp.
Leptolyngbya sp.1
Leptolyngbya sp.2
Leptolyngbya sp.3
Leptolyngbya sp.4
Oscillatoria sp.1
Oscillatoria sp.2
Phormidium autumnale
Phormidium sp.1
Phormidium sp.2
Phormidium sp.3
Phormidium sp.4
Pseudoanabaena sp.1
Pseudoanabaena sp.2
Number of species
Bacillariophyceae
Achnanthes exigua Grunow in Cleve and Grunow
Achnanthes minutissima Kützing
Gomphonema sp. Ehrenberg
Hantzschia amphioxys (Ehren.) Grunow in Cleve and Grunow
Navicula contenta Grunow in Van Heurck
Navicula mutica Kützing
Navicula mutica var. cohnii
Navicula pelliculosa (Brébisson ex Kützing) Hilse
Nitzschia palea (Kützing) W. Smith
Surirella minuta Brébisson in Kützing
Abandoned field
Pasture
Vineyard
March
May
March
May
March
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
May
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
9
9
+
+
12
8
4
1
3
+
+
+
+
+
2
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
7
7
3
3
2
3
+
Number of species
4
4
Xanthophyceae
Chloridella cystiformis Pascher
Heterococcus sp. Chodat
Xanthonema montanum (Vischer) Silva
+
+
+
+
+
Number of species
2
1
1
1
0
+
+
+
+
Chlamydophyceae
Chlamydomonas callunae Ettl
Chlamydomonas oblongella Lund
Chlamydomonas sp.2
Chlamydomonas sp.4
Chlamydomonas sp.5
Chlamydopodium fusiforme (Lee and Bold) Ettl and Komárek
Chlorococcum novae-angliae Archibald and Bold
Chlorococcum schwarzii Ettl and Gärtner
Chlorococcum ellipsoideum Deason and Bold
Chlorococcum infusionum (Schrank) Meneghini
March
+
+
Number of species
May
+
+
+
+
+
Eustigmatophyceae
Eustigmatos magnus (J.B. Petersen) Hibberd
Pseudocharaciopsis ovalis (Chodat) Hibberd
Vischeria helvetica (Vischer and Pascher) Hibberd
Corn field
+
+
2
+
0
1
+
+
1
1
+
+
1
1
1
1
2
0
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
S. Zancan et al. / Agriculture, Ecosystems and Environment 112 (2006) 1–12
7
Table 3 (Continued )
Abandoned field
Pasture
March
March
May
+
+
+
+
+
May
Chlorococcum sp.2
Chlorococcum sp.3
Nautococcus solutus Archibald
Neospongiococcum macropyrenoidosum Deason and Cox
Sphaerellocystis sp. Ettl
Tetracystis compacta Schwarz
Tetracystis pulchra Brown and Bold
Tetracystis sp. Brown and Bold
Chlorococcacean alga sp.1
Number of species
Chlorophyceae
Bracteacoccus minor (Chodat) Petrova
Chlorella mirabilis Andreeva
Chlorella ellipsoidea Gerneck
Chlorella emersonii Shihira and Krauss
Chlorella homoshaera Skuja
Chlorella keslerii Fott and Nováková
Chlorella luteoviridis Chodat in Conrad and Kufferath
Chlorella minutissima Fott and Novàkovà
Chlorella saccharophila (Krüger) Migula
Chlorella vulgaris Beijerinck
Chlorosarcinopsis delicata S. Watanabe
Chlorosarcinopsis gelatinosa Chantanachat and Bold
Chlorosarcinopsis minor (Gerneck) Herndon
Chlorosarcinopsis sp. Herndon
Desmococcus olivaceus (Pers. Ex. Ach.) Laundon
Dictyosphaerium chlorelloides (Naumann) Kom. and Perman
Elliptochloris subsphaerica (Reisigl) Ettl and Gärtner
Gloeocystis vesiculosa Nägeli
Leptosira sp. Borzi
Muriella decolor Vischer
Muriella terrestris J.B. Petersen
Muriella zofingiensis (Dönz) Hindák
Neochloris sp. Starr
Protosiphon botryoides Klebs
Pseudochlorella pyrenoidosa (Zeitler) Lund
Pseudococcomyxa simplex (Mainx) Fott
Scenedesmus soli
Scotiellopsis sp.
Thelesphaera alpina Pascher
Vineyard
March
Corn field
May
March
May
+
+
+
4
0
3
+
+
+
+
+
+
6
6
3
4
6
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Number of species
9
10
12
15
4
6
10
10
Klebsormidiophyceae
Klebsormidium dissectum (Gay) Ettl and Gärtner
Klebsormidium flaccidum (Kützing) Silva, Mattox and Blackwell
Stichococcus bacillaris Nägeli
+
+
+
+
+
+
+
+
+
+
+
+
Number of species
2
3
1
2
2
2
0
1
Ulvophyceae
Pseudendoclonium basiliense Vischer
+
+
Number of species
1
1
0
0
0
0
0
0
Zignematophyceae
Cylindrocystis brebissonii var. minor W. West and G. S. West
+
Number of species
1
0
0
0
0
0
0
0
Total number of species
33
45
29
37
50
40
21
26
17
20
31
24
+
8
S. Zancan et al. / Agriculture, Ecosystems and Environment 112 (2006) 1–12
March and 16 8C in May). Soil temperature and water
content differed at the two sampling times (Table 1).
3.2. Algal taxa and communities
In all, 92 algal taxa (belonging to 50 genera) were
recorded in the two sets of topsoil (0–2 cm) samples from
the four sites. Chlorophyta (53 taxa) were by far the most
diverse group (29 Chlorophyceae, 19 Chlamydophyceae, 3
Klebsormidiophyceae, 1 Ulvophyceae and 1 Zignematophyceae), followed by Cyanophyceae (23 taxa), Bacillariophyceae (10 taxa), Xanthophyceae (3 taxa) and
Eustigmatophyceae (3 taxa).
The algal taxa and the sites where they were recorded are
listed in Table 3. The abandoned field and pasture supported
richer algal communities. There was a wealth of green algal
species in all eight samples, while the abandoned field and
pasture had more species of cyanobacteria and diatoms than
in the vineyard or corn field.
3.3. Density of soil algae
As shown in Fig. 3, the total average density of soil algae
varied from 99 103 to 1483 103 algal cells g1 soil
(d.w.). Higher densities, above 850 103, were recorded in
the abandoned field and pasture, by comparison with the
vineyard and corn field, under 200 103. Total algal density
was significantly higher in March than in May in both the
abandoned field and the pasture, whereas the time-related
differences were not significant for the other two sites.
The distribution of CH + X + E (Fig. 4) closely reflected
the total densities because this group accounted for a very
high relative density in all samples analysed. Cyanobacteria
were equally abundant in the four samples from the
abandoned field and pasture (>270 103 g1 soil d.w.),
while much lower densities were recorded in the corn field
(<25 103 g1 soil d.w.) and particularly in the vineyard,
Fig. 3. Total abundance of algae in abandoned field, pasture, vineyard and
corn field at the two sampling times (ab: abandoned field; pa: pasture; vi:
vineyard; co: corn field).
Fig. 4. Abundance of the CH + X + E algal group (green algae + Xanthophycea + Eustigmatophycea) in abandoned field, pasture, vineyard and corn
field at the two sampling times (ab: abandoned field; pa: pasture; vi:
vineyard; co: corn field).
where cyanobacteria were virtually absent (<3 103 g1
soil d.w.) (Fig. 5). The diatoms were most abundant in the
March sample from the pasture (>120 103 g1 soil d.w.)
and most scarce in the May sample from the abandoned field
and in all four vineyard and corn field samples
(<12 103 g1 soil d.w.) (Fig. 6).
3.4. Relative contributions of main algae groups
In the abandoned field, CH + X + E algae contributed
substantially to the total density of the algal community (76%
in March, 73% in May), while cyanobacteria accounted for
20% in March and 26% in May and diatoms were more rare (4
and 1%) (Fig. 7). In the pasture, the proportion of CH + X + E
was 67% in March and 58% in May, cyanobacteria amounted
to 23 and 34%, diatoms again revealed the lowest density (10
and 8%). In the vineyard, the contribution of CH + X + E was
substantial, with 90% in March and 95% in May, while
Fig. 5. Abundance of the CY (Cyanobacteria) algal group in abandoned
field, pasture, vineyard and corn field at the two sampling times (ab:
abandoned field; pa: pasture; vi: vineyard; co: corn field).
S. Zancan et al. / Agriculture, Ecosystems and Environment 112 (2006) 1–12
9
Fig. 6. Abundance of the D (Bacillariophyceae) algal group in abandoned
field, pasture, vineyard and corn field at the two sampling times (ab:
abandoned field; pa: pasture; vi: vineyard; co: corn field).
cyanobacteria were nearly absent (1.5 and 2%) and the
proportion of diatoms was 8.5 and 4%. In the corn field,
CH + X + E algae were again the dominant group, with 72%
in March and 75% in May, as opposed to 25 and 20% of
cyanobacteria, and 3 and 5% of diatoms.
The CH + X + E algal group was dominant in all samples
analysed, marginally less so in the pasture (where diatoms
and cyanobacteria reached the highest relative density
values), but even more so in the vineyard, where
cyanobacteria were almost absent (Fig. 7).
Fig. 8 shows the dendrogram obtained from cluster
analysis based on the species presence/absence matrix. Four
groups are distinguishable, each comprising the two
samples from one site. The greatest similarity between
the two samples from a given site came from the pasture (37
taxa in March, 40 in May, 27 taxa in common), while the
abandoned field revealed the greatest diversity (33 taxa in
March, 29 in May, 19 taxa in common). The following
clusters emerged: cluster 1, the two corn field samples;
cluster 2, the two vineyard samples; cluster 3, the four
samples from the pasture and the abandoned field. The two
corn field samples (characterised by few species and six
exclusive taxa) showed the greatest scatter in terms of
species composition.
Fig. 7. Relative contribute of algae main groups to total abundance in
abandoned field, pasture, vineyard and corn field at the two sampling times
(CH + X + E: green algae + xanthophycean algae + eustigmetophycean
algae; CY: cyanobacteria; D: diatoms).
3.5. Algae community and soil variables
The uncorrelated environmental soil variables identified
by Pearson’s correlation matrix that were used for variance
analysis were: topsoil temperature, rainfall in the 20 days
prior to sampling, pH, N, P, organic carbon, C/N, K, Na, Ca,
Cu, Cd and Zn, and proportion of clay, silt and sand.
The two principal axes account for 77% of the variation.
The vineyard is distinguishable from the other sites for its
higher soil moisture, lower pH values, higher organic C and
higher K, Ca, Cu and Cd soil content (Fig. 9).
The first two components of the variance analysis based on
algal community variables justify 66% of the variance in the
data. This analysis enabled the corn field samples to be
grouped together with the vineyard samples, since they had in
common a limited number of cyanophytes and diatoms, a
lower total density of the algal community and a higher density
in May than in March. The intra-site differences were greatest
in the abandoned field, and smallest in the corn field (Fig. 10).
Fig. 8. Dendrogram of complete link pair-group cluster analysis of algal taxa based on Jaccard’s index.
10
S. Zancan et al. / Agriculture, Ecosystems and Environment 112 (2006) 1–12
Fig. 9. Ordination diagram based on environmental variables: topsoil
temperature, rainfall in the 20 days prior to sampling, pH, N, P, organic
carbon, C/N, K, Na, Ca, Cu, Cd and Zn, and proportion of clay, silt and sand
(ab1: abandoned field March sample; ab2: abandoned field May sample;
pa1: pasture March sample; pa2: pasture May sample; vi1: vineyard March
sample; vi2: vineyard May sample; co1: corn field March sample; co2: corn
field May sample).
4. Discussion
Soil pH is an important factor in determining the
composition of algal communities. At all sites, the soil pH
varied little, between 7.28 and 8.03, so all the main algal
groups were represented. Cyanobacteria are unable to
survive in acidic conditions (Brock, 1973), while abundant
and diverse green algae have been recorded in acid soils
(Lukešová and Hoffmann, 1995). Neutral conditions, such
as those recorded in the study, support the growth of algal
communities representing all the main taxonomic groups
(Metting, 1981; Lukešová, 2001).
At all four sites, green algae were represented with the
largest number of species, followed by blue-green algae and
diatoms. Xanthophyceae and Eustigmatophyceae were found
in all samples, but were only represented by one or two species.
Fig. 10. Ordination diagram based on algal community variables: total algal
density, CH + X + E, CY and D density, number of taxa identified belonging
to different algal classes (ab1: abandoned field March sample; ab2: abandoned field May sample; pa1: pasture March sample; pa2: pasture May
sample; vi1: vineyard March sample; vi2: vineyard May sample; co1: corn
field March sample; co2: corn field May sample).
As in other studies on soil algae of European temperate
non-forest ecosystems where species were identified
(Lukešová, 1993; Lukešová and Hoffmann, 1996; Neustupa,
2001; Lukešová, 2001), a great variety of species was
recorded at each site. Though critical comparisons with
published data are often hindered by different experimental
conditions and quantitation methods, often linked to the
difficult and tedious nature of species identification (Round,
1981; Stellmacher and Reisser, 1999), our findings confirm
that Chlorella minutissima Fott and Novàkovà, Klebsormidium flaccidum (Kützing) Silva, Mattox and Blackwell
(Fig. 2a), Stichococcus bacillaris Nägeli, Xanthonema
montanum (Vischer) Silva (Fig. 2f), Hantzschia amphioxys
(Ehrenberg) Grunow in Cleve and Grunow (Fig. 2d),
Navicula pelliculosa (Brébisson ex Kützing) Hilse, Anabaena sp. and Nostoc sp1 can be considered cosmopolitan
and widespread in different soils (Ettl and Gärtner, 1995). In
our case, they were found widely distributed (in at least six
of the eight soil samples analysed), but probably they were
not the quantitatively dominant species in our soil algal
communities. As in Starks and Shubert (1981), many algal
species seemed to compete equally and none were able to
dominate the system in quantitative terms.
Pseudendoclonium basiliense is one of the two exclusive
to the abandoned field algal taxa. It is found both in
freshwater and in soil habitats; in the soil, it was reported
with low frequency in an intensively cultivated field and in
abandoned fields of South Boemia (Lukešová, 1993).
The pasture and abandoned field, in particular, supported
numerous cyanobacterial populations, with many nonheterocystous species. Cylindrospermum sp. (Fig. 2b) and
Scytonema sp. were recorded among the heterocystous
species. Conversely, Scytonema was only found in disturbed
soils treated with commercial fertiliser (King and Ward,
1977). The vineyard and corn field soils were less rich in
species, due to a fewer species being identified in all the
main groups, and the cyanobacteria in particular.
Algae were abundant in all the sites, always more than
100 103 cells/g of soil (d.w.), with peaks of about
1500 103 cells/g of soil (d.w.). Favourable temperature
and pH, as well as adequate light and abundant essential
mineral nutrients would seem to be important in favouring
such high cell concentrations (Ruble and Davis, 1988). The
site least rich in algae was the corn field. Algae probably
respond rapidly and positively to the regular use of
fertilisers, their abundance and diversity increasing consistently with chemical fertiliser application. But lengthy
periods of intense fertilisation lead to a reduction in species
diversity and a suppression of blue-green algae development
(Kuzyakhmetov, 1998a). This seems the likely reason for the
low abundance and low diversity of the algal communities
and the almost complete absence of Cyanobacteria in the
corn field, which had been treated with high-input, twiceyearly mineral fertilisations for many years (one before and
one at the time of seeding). Kuzyakhmetov (1998a)
attributed a low abundance of algae in arable lands to the
S. Zancan et al. / Agriculture, Ecosystems and Environment 112 (2006) 1–12
peculiar environment conditions induced by the periodical
loosening and greater dryness of the topsoil due to farming.
These conditions negatively affect many algal species,
especially some blue-green algae (Kuzyakhmetov, 1998a),
e.g. Nostoc sp., Phormidium autumnale, Phormidium sp.
and Pseudoanabaena sp.
In addition to fertiliser, the corn field was also treated with
a pre-emergent herbicide between the two sample collections.
Pesticides are known to affect algae in vitro and in vivo, with
algal species varying considerably in their sensitivity to
various pesticide concentrations (McCann and Cullimore,
1979; Megharaj et al., 1999; Mostafa and Helling, 2002). Preemergent herbicides would influence the range of genera and
the number of algal cells present at any given time (McCann
and Cullimore, 1979), and cyanobacteria in particular
(Metting and Rayburn, 1979). This herbicide did not induce
a decline in the abundance of the algae, however, possibly due
to soil heating and not limiting moisture conditions balancing
the negative effects of the pesticide. In fact, the algae were
slightly more abundant in May than in March.
The samples of vineyard showed a relatively low
abundance of algae and number of species, with particularly
low counts for cyanobacteria. The algal community structure
may be modified in this case by a combination of fungicide
applications and tillage. It has been demonstrated that
dithiocarbamate-based fungicides, as used in our case,
interfere with the formation and germination of Cylindrospermum sp. akinetes (Panigrahy and Padhy, 2000). Akinetes
formation and germination are particular important phases for
overcoming the unfavourable seasons. Massive use of
fungicide may take part in the destruction of the cyanobacterial soil component. Antiperonospora cupric compounds
(particularly copper oxychloride) were used at the vineyard
and this was reflected in the higher copper concentrations
found in the vineyard soil samples (53 mg g1 soil d.w.).
Copper compounds have negative effects on the structure and
function of algal soil communities; and copper sulphate is
more toxic for green algae than oxychloride. Low doses of
copper compounds can stimulate the development of tolerant
algal species, however (Kuzyakhmetov, 1998b).
The abandoned field and pasture soil samples had an
abundance of algae and were characterised by similar flora.
These two sites had not been tilled or treated with pesticides
or chemical fertilisers for many years. The large number of
algae recorded below the chemically-unfertilised, permanent grass cover may be due to inorganic nutrients returning
to the surface layers of the soils and becoming available to
surface-dwelling algae, by the decay and mineralization of
dead above-ground vegetation (Broady, 1979). In fact, the
algae communities of pasture are significantly richer than
those of hay fields, where biomass is periodically removed
(Kuzyakhmetov, 1998a). In the abandoned field, and in the
pasture (where vegetation was much more developed in May
than in March), the algal abundance was lower in the second
sample, probably because of a smallest amount of light
being transmitted through the canopy.
11
Our data confirm the considerable effect of agricultural
practices on the structure of soil algal communities
(Table 3). Earthworms apparently respond in much the
same way (Paoletti et al., 1988). The corn field could be
grouped with the vineyard in term of total algal density, main
group density, and number of taxa identified belonging to
different algal classes. These two sites were characterized by
low algal abundance (<2 105 cells/g of dry weight soil),
low number of taxa (<25 taxa). The limited abundance and
variety of species was particularly evident for cyanobacteria.
The dendrogram obtained from cluster analysis based on the
presence/absence matrix enabled the pasture to be grouped
together with the abandoned field, both characterised by a
large number of identified taxa (29–41) and a relative large
number of cyanobacteria and diatoms.
5. Conclusions
Less disturbed sites support more diverse and abundant
algal communities than sites submitted to the intensive
ploughing, planting, herbicide and fungicide applications,
fertilisation and harvesting associated with farming. Soil
algae community structures are affected more by soil usage
than by physico-chemical parameters. Cyanobacteria show
the most evident response in the different agro-ecosystems,
and consequently seem to be the most suitable group to
adopt as a soil bioindicator of land use. As suggested by
Paoletti (1999), however, there is a general need to improve
our knowledge of micro-organisms, including algae, to
better appreciate the many benefits that humans derive from
their existence. Further information of this kind, using
standard quantification methods and precise identification
procedures, should therefore be obtained in order to draw
general conclusion about the potential role of soil algae as
bioindicators.
Acknowledgement
We thank Prof. A. Squartini for the assistance during the
fieldwork.
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