Romanian approaches on mycgorrhiza in the frame of European researches, ed. [615452]

Romanian approaches on mycgorrhiza in the frame of European researches, ed.
T.E. Sesan, Editura Universitatiii B ucuresti, ISBN 978 -973-737-901-6

Effects of bioactive mulch system on , water stable aggregates , mycorrhizal
propagules and glomalin from a cambic chernozem soil.

F. Oancea1a, Florica Constantinescua, Tatiana Eugenia Sesanb, D. Manolec, Sorina Dinua, Oana Sicuiaa
a- Research – Deve lopment Institute for Plant Protection, Bd. Ion Ionescu de la Brad nr. 8 sect.1, Buchares t 013813
b- University Bucharest, Faculty of Biology Aleea Portocalilor , No. 1, sect. 6, Bucuresti , 060101
c- SC Sportagra Srl , Str. Mare pozitia 38 -10, 907030 Amzace a, Constanța county

Abstract
We developed a conservative agriculture system based on a "bioactive mulch", resulted from mulched
winter cover crop , cover crop residues being treated with microorganisms antagonist to soil born phytopatogens and
seed of mai n crop being inoculated with diazotroph plant growth promoting rhizobacteria.
We evaluated the effects of this conservative agriculture system on water stable aggregates (WSA),
arbuscular mycorrhizal fungi (AMF) propagule s and glomalin (easy extractab le and total) . The field experiment
was done a cambic chernozem (Amzacea, Dobroudja), using hairy vetch as a cover crop and sunflower as a main
crop. The experiment shown that bioactive mulch agricultural system increase water stable aggregates and glomalin
(total and easily extractable) on soil , especially in the shallowest (0 –5 cm) layer of soil. Hairy vetch as a cover crop,
fungal antagonist (Trichoderma viride Td50) applied to mulched hairy vetch and sunflower plant developed from
seed inoculated with Azospirillum brasilense SF12, slightly increase the AMF propagule numbers. Bacterial
antagonist ( B. amyloliquefaciens B165) used on the bioactive mulch system, as treatment of cover crop mulch ,
combined with sunflower plant developed from seed inoculated wi th Azospirillum brasilense SF12 , have a more
significant effect on increase of AMF propagule on soil.
The results prove that conservative agricultural system based on bioactive mulch increase the activity of the
AMF on cambic chernozem from Dobroudja are a.

Key words: bioactive mulch, glomalin, water stable aggregates, AMF propagule.

Introduction
Converting winter cover crop (WCC) into a mulch laying down on top soil appear to be
one of the most promising conservation practice in agriculture [10,13,35 ,39]. Winter cover crops
can maintain or increase soil C and N [21,22,31 ] and these effects are largely responsible for the
changes in physical properties associated with their use [12] . Maintenance of a layer of crop
residue over the soil surface helps to protect from raindrop impact and cycles of freezing –
thawing and drying –wetting [4,33,46] .
In the mean time l arge amounts of crop residue on the soil surface may particularly favor
pathogens that survive between crops in th e infected/infested residue [3,2 9]. Crop residues lying
on the surface reduce soil temperatures. Lower soil temperatures caused by plant residues
retarded seed germination and crop growth ( including a delay of root system development ),
promote root disease s and finally reduced crop yield s [1,17,38] .
In order to counter act these negative side effects of cover crop mulches w e proposed : (i)
the application on crop residues laying on soil of an antagonist to the soil born phytopat hogens
and (ii) the inoculation with diazotroph plant growth promoting rhizobacteria (PGPR) of the seed
of cash crop established on antagonist treated mulches [27]. Antagonist to plant pathogen (like
fungi from genus Trichoderma or bacteria from genus Bacillus ) are intended to reduce the
primary inoculum for both f oliar infecting and root infecting soil born phytopathogens .

1 corresponding author, e -mail: florin.oancea@icdpp.ro

Diazotroph PGPR (like Azospirillum ) aim to promote t he development of cash crop seedling and
to improve the nutrition of the plant with a slow er developing root system.
We called this system , which include antagonist (to phytopathogens) application to WCC
mulch and diazotroph PGPR inoculation to cash crop seeds , "bioactive mulch ". Due to the use
of highly competitive microbial strains t his approach could have negative influence on soil
microbial community , including on mycorrhizal fungi. Soil microorganisms (and especially
mycorrhizal fungi) are related to t he positive effects of WCC mulch on soil [5,14,34] It has
been demonstrated that some antifungal strains of both Trichoderma or Bacillus genera may
decrease AMF activity [15,24,47] .
This study was designed to evaluated the effects of the "bioactive mulch" conservative
agriculture system (and especially of antagonistic microorganism from Bacillus and
Trichoderma genera) on several soil indic ators, related to soil quality and mycorrhizal fungi
activity, like water stable aggregates (WSA) , arbuscular mycorrhizal fungi (AMF) propagule s
and glomal in (easy extractable and total) .

Material and method s.
Experimental design . Experiment were done in a field experiment, on a cambic
chernozem (Amzacea, Dobroudja). Hairy vetch ( Vicia villosa cv. Hungvillosa) was used as a
cover crop. It was seeded into corn stubble on beginning of October, on a seeding rate of
120…125 seed.m-2, corresponding to 38 k g.ha-1. Hairy vetch seeds were inoculated with
Rhizobium leguminosarum bv. viciae Mz269 (NCAIM 001362 ). Hairy vetch cover crops were
killed by rolling with a roller -crimper and concomitant application of glyphosate (N –
(phosphonomythyl) glycine) at 1.1 kg a.i. ha-1 in the spring, 2 days before establishing cash
crop. Sunflower (Helianthus annuu s cv. Rumbasol) was used as cash crop and sown on April 3rd,
at a row width of 0.7 m. Target plant population was within the range 5.5 –6.5 plants .m-2 and the
seedin g rate was 4,3 kg ha-1. On all treatment sunflower seeds were drilled at 5 cm depth into
soil by a pneumatic direct planting machine. The sunflower seeds used on all no-till treatments
were inoculated with 106 ufc.g-1 Azospirillum brasilense SF12 . On bioa ctive mulch treatment
plant residues covering the soil was sprayed with a liquid suspe nsion of antagonist to soil born
phytopathogens , Trichoderma viride Td50 NCAIM 001358 (2.5.106 spores.ml-1) or Bacillus
amyloliquefaciens B165 NCAIM B001363 (107 spore.ml-1). The suspension was applied on a
volume equivalent to 700 l.ha -1 using a “Solo -432” (Solo, Newport, VA, USA), motor -driven
knapsack sprayer with a tank capacity of 20 l and a spray lance length of 0.7 m. The experiment
was organiz ed in a Latin square design , including 4 treatments in 4 repetitions : V 1 – intensive
management, no cover crop, deep plo ughing with soil inversion on 25 cm on the beginning of
October, surface tillage with a disc harrow 2 days before sunflower planting , weed control by
mechanic al weeding ; V 2 – cover crop mulch , seed inoculated with 106 ufc.g-1 Azospirillum
brasilense SF12 , no other weed control than mulch cover ; V3 – cover crop mulch treated with 107
spores.ml-1 B. amyloliquefaciens B165 , seed inoculated with 106 ufc.g-1 Azosp irillum brasilense
SF12 , no other weed con trol than mulch cover ; V4 – cover crop mulch treated with 2.5.106
spores.ml-1 T. viride Td50, seed inoculated with 106 ufc.g-1 Azospirillum brasilense SF12, no
other weed control than mulch cover. Each block of a r epetition consist of a plot of 5 m wide and
10 m long.
Soil sampling . Soil samples were collected before sunflower harvest, in 22 September,
after a rain event which ensured uniform water content and as close to field capacity as possible.
Bulk soil used for aggregate analyses was collected from the 0 to 5 cm and 5 to 10 cm depth of
non-wheel traffic inter-rows by compositing four, shallow excavations (0.2 by 0.1 m by 0.01 m
deep) from each plot. Two soil (sub) samples per plot to 30 -cm depth were taken from the
shallow excavation with a hand auger sampler ( Eijkelkamp Agrisearch Equipment BV,
Giesbeek, The Netherlands) for soil glomalin assay . The cores of soil resulted from sampler were

cut to obtain 10 to 20, and 20 to 30 cm subsamples. All soil samples were stored in zip lock
plastic bags and kept on 4 șC till analyses were performed .
Water stable aggregate assay . Water -stable aggregates were measured by wet sieving on
stacked sieves in a manner similar to that described by Arshad et al . [2]. First, the dried soil
samples were passed though a 6 mm screen and then through 2 mm screen. Five grams of soil
passed through 2 mm screen were placed on a 1000 µm sieve. Stacked under the 1000 µm sieve
was a 250, a 125, and a 53 µm sieve, spaced about 1 cm apart vertically. The sieve set was
immersed in deionized water until the soil sample was completely covered, then immediately
sieved for 3 min at 20 cycles per minute. The length of stroke was 1.3 cm. This stroke and
duration were sufficient to clear the screen s of slaked soil, leaving only separated aggregates too
large to pass through each sieve. The weight of soil retained on each sieve was determined after
drying at 40ș C. The proportion of water -stable aggregates (WSA) in each size fraction (WSA i)
was calcul ated from Eq. (1) :

Sandi MoistSoilSand AggWSAi i
i
) 1(
(1)
where i is the ith size fraction, Agg is the oven -dry mass of water -stable aggregates collected on
each sieve, Sand is the oven -dry mass of sand collected on each s ieve, Soil is the oven -dry mass
total 2 – to 6-mm aggregates sieved, and Moist is the gravimetric moisture content.
The MWD of aggregates was calculated from Eq. (2):
 i iWSAX MWD
(2)
where i is the ith size fraction, and X is the mean diameter of each size f raction, based on the
mean intersieve size. Water stable a ggregate (WSA) is the sand -free, water -stable aggregate
mass. Sand is the oven -dry mass of sand collected on each sieve .
Arbuscular mycorrhizal fungi (AMF) propagule assay . The number of AMF was
estimated by direct counts of sporocarps and spores occurring in the soil samples and extracted
by wet sieving and sucrose densit y gradient centrifugation [28] . The assay included passage of 50
g of air -dried field soil through 1,000 -, 500 -, 125 -, and 32 -µm sieves. The 1,000 -µm sieve was
checked for spores adjacent to or inside roots, while the 500 -µm sieve was checked for large
spores, spore clusters, and sporocarps. The contents of the 125 – and 32 -µm sieves were layered
onto a 70% water -sucrose solution (wt /vol) gradient and centrifuged at 900 x g for 2 min. The
resulting supernatant was passed through the 32-µm sieve, washed with tap water, and
transferred to petri dishes. Spores, spore clusters and sporocarps obtained from all sieves were
counted by using a dissecting microscope at a magnification of up to x90. Thereafter, 50 to 70%
of them were mounted on slides with polyvinyl -lactic acid -glycerol [20] . Only the healthy –
looking spores were mounted. The spores were examined under a stereomicroscope (SZX10,
Olympus) at a magnification of up to x400. Only 60 to 75% of the spores mounted on slides
could be identified to the species level or attributed to a specific morphospecies; the rest
consisted mostly of old and decaying spores with missing clear features. (International Culture
Collection of Arbuscular Endomycorrhizal Fungi [ http://invam.caf.wvu.edu/Myc_Info/
Taxonomy/species.htm ]). The abundance of spores from all AMF species together w as
determined for each sample and expressed as the number of AMF spores per gram of soil for the
field samples. Also for each sample from the field treatments, the number of spores belonging to
the different AMF species discerned also was determined.
Glomalin content assay . The procedure used for assay of glomalin -related soil protein
(GRSP), easy extractable and total , is presented in fig.1. This is an adaption of methods
described by Nicholson and Wrig ht [26] and Wright et al. [41] . The procedure was d one for 1 g
of soil sample. Each sample was initially extracted by autoclaving , at 121șC for 30 min , of a

mixture of 1 g of soil and 8 ml sodium citrate buffer 20 mM, pH 7.0 . After cooling and
centrifugation for 10 min at 10,000 g was separated the superna tant (containing easy extractable
GRSP) and a residue. The residue was extracted with NaOH for 1 h, at room temperature. The
supernatant was removed by centrifugation at 10,000 g and on remaining residue two additional
sequential 1 h extractions were perfor med, using each time 8 ml of 100 mM sodium
pyrophosphate, pH 9.0 and autoclaving at 121ș C for 1 h . The supernatant s separated by
centrifugation at 10,000 g from each extraction cycle were combined and the resulting final
volume (containing recalcitrant GRS P) was measured. Proteins in the supernatants were
analyzed using a Bradford protein assay with bovine serum albumin as standard. Total glomalin /
GRSP was calculated as the sum of easy extractable + recalcitrant.
Soil
Easily extractable
GRSP poolSediment
Mixture of humic
and fulvic acid
Sediment
Recalcitrant
glomalinSoil
residueHumic acid
precipitateFulvic acid
solutionextraction 20 mM citrate buffer
121șC, 30 min, pH 7.0
extraction NaOH 1 M,
25șC, 1 h
Soluble in
weak acidprecipitation by neutralization
with acid extraction 100 mM pyrophosphate
121șC,1h, pH 9.0

Fig. 1 . Sch ematic representation of the procedure used for the extraction of glomalin -related soil
protein (GRSP easy extractable and recalcitrant ) from soil of different treatments. Extraction procedure
adapted with modificatio ns from Nicholson and Wright [26] and W right et al. [41].
Statistical analysis . The mean weight diameter was computed for each water -stable
aggregate measurement by summing the product of each fraction times its mean inter -sieve size
[2]. A separate analysis was performed on the field plots u sing a mixed model with treatment
and sample depth (0 –5 cm, 5 –10 cm). Differences among the treatments and within treatments
were tested by one -way analysis of variance, after Bartlett’s test of equal variances was satisfied,
using Statistica 9.0 ( StatSoft , Tulsa, Oklahoma, USA) . Results were considered significantly
different at the P < 0.05 level.

Results and discussions
The comparison of the mean weight diameter of aggregates in the field plot experiment
presented in fig. 2 show a significant differ ence between intensive crop control and conservative
treatments . The increase of the mean weight diameter is present on both depth s of mulch covered
treatments. The treatment with microorganisms antagonist to soil born phytopathogens ( T. viride
Td50 and B. amyloliquefaciens B165) did not affect the mean weight diameter of water stable
aggregates. More, the use of these microorganism into b ioactive mulch agricultural system
slightly increase water stable aggregates compared with WCC mulch alone.

Fig. 2. Comparison of the mean weight diameter of aggregates in the field plot experiment. Least –
square means from mixed model with error bars showing the standard error of each mean. Main effect of
treatment significant at p < 0.01.

Soil aggregate stability is the result of complex interactions among biological, chemical,
and physical processes in the soil [23]. The concept of aggregate stability depends on both the
forces that bind particles together and the nature and magnitude of the disruptive stress .
Aggre gate stability decli nes rapidly in soil planted to a clean tilled and maintained crop,
ploughing and mechanical weeding disrupting soil aggregate. Also clean soil expose top soil
aggregate to various disruptive atmospheric stress (e.g. rain splash). Mulch covering the soil
promote aggregate stability because is offering a protection from raindrop impact and cycles of
freezing –thawing and drying –wetting [4,33,46] . Only on intensive control the mean weight
diameter of water stable aggregates is smaller on 0 -5 cm depth comparing with 5 -10 cm depth
(Fig. 2 ), and this is due also to the soil exposure on intensive, clean soil maintained crop, which
finnaly lead to a reduction on dimension of water stable aggregate. O n mulch covered treatment
the mean weight diam eter of water stable aggre gate are statistically similar for both depths
analyze d (0-5 cm, 5 -10 cm) and this is showing the existence of similar condition on upper soil
horizons .
Another explanation for increase of WSA , beside physical protection from di sruptive
stress, is the enhancement of microbi al activity by the mulches; mulches are buffering
atmospheric variation on the soil surface , offering more stable conditions . Arbuscular
mycorrhizal (AM) fungi are a mong the microorganism the most involved in water -stable
aggregation of soil near the surface [5,30,40]; thus we investigate d also the effect of bioactive
mulch on arbuscular mycorrhizal fungi (AMF) propagules .
Analysis of AMF species from the experimental site shown that these belonged to the
genera Glomus and Scutellospora and were: Glomus badium Oehl, Redecker & Sieverd., Glomus
etunicatum (Becker & Gerdemann), Glomus geosporum (Nicol. & Gerd.) Walker, Glomus
mosseae (Nicol. & Gerd.) Gerdemann and Trappe, Glomus sinuosum (Gerdemann and Bakshi)

Almeida and Schenck (Basionym: Sclerocystis sinuosa Gerdemann and Bakshi), Scutellospora
calospora (Nicol. & Gerd.) Walker & Sanders.
The abundance of total intact free AMF spores (SA) and small spored Glomus (SS)
species in the field experimental plot is presented in Figure 3. This abundance is rather medium
and t he differences among to tal numbers of AMF spores in different treatments were mostly due
to spores in the smallest size class (under 100 μm). Both the small spore fraction and the total
number of spores were affected by different treatments ( P<0.1, Fig. 3).

Fig. 3. AM s pore counts in field soils after different treatment . SA – total intact free AMF spores; SS –
small spores Glomus (under 100 μm ).

Hairy vetch as a cover crop, fungal antagonist ( Trichoderma viride Td50) applied to
mulched hairy vetch and sunflower plant developed from seed inoculated with Azospirillum
brasil ense SF12, slightly increase the AMF propagule numbers. Bacterial antagonist ( B.
amyloliquefaciens B165) used on the bioactive mulch system, as treatment of cover crop mulch,
combined with sunflower plant developed from seed inoculated with Azospirillum brasilense
SF12, have a more significant effect on increase of AMF propagule on soil.
It has been demonstrated that some antifungal strains of both Trichoderma or Bacillus
genera may decrease AMF activity [15,24,47] . But there are studies showing a stimu latory effect
of Trichoderma or Bacillus antifungal strains on AM fungi [11,25,37] . B. amyloliquefaciens
B165 and T. viride Td50 are among the antifungal strains which are not inhibiting the activity of
AMF fungi. It seem that B. amyloliquefaciens B165 pre sent characteristics of mycorrhizal helper
(because is increasing significantly the AMF propagules on soil on the given experimental
conditions), but this need further investigation.
AM fungi produce large amounts of an insoluble glue -like substance, glo malin on
hyphae [9,42] . Glomalin is an abundant component of soil organic matter and has been linked to
aggregate stability [26,43,44,45] . Our investigation targeted also on the effect of bioactive mulch
on soil glomalin.

The results of analysis of glomal in on different depths and treat ment are presented in
Table 1. The increase in glomalin -related soil protein (GRSP, Bradford reactive soil protein
extracted according to F ig.1) concentration s on mulched treatments (and especially on
biomulched one) reflect an increase in AMF biomass / AMF activity. The easily extractable and
total glomalin contents were quite low , but similar to those reported in other studies carried out
in soils containing calcium carbonate und er similar climatic conditions [30,45] . The f act that the
total GRSP increased more than the easily extractable Bradford -reactive soil protein following
mulching seems to contradict the initial idea that the easily extractable GRSP relates to recent
depo sits [30] . Here we should consider also the hum ification potential of cover crop residue s,
which could lead to a faster complexation of GRSP with the polyphenolics matrix of humic acid.
Table 1. Easy extractable (E) and total (T) g lomalin -related soil protein (GRSP) concentration s (mg
g–1 soil) on different soil depths and treatmenta.
Treatment Depth of soil sample
0-5 cm 5-10 cm 10-20 cm 20-30 cm
T-
GRSP E-
GRSP T-
GRSP E-
GRSP T-
GRSP E-
GRSP T-
GRSP E-
GRSP
Control, intensive management, 3,47c 1,82b 3,62c 1,94b 2,68c 1,52b 2,24b 1,36b
Cover crop mulc h 4,72b 2,63a 4,86ab 2,57a 3,42b 1,76a 3,16a 1,62a
Bioactive mulch, B165 + SF12 5,74a 2,82a 5,48a 2,68a 3,87a 1,82a 3,08a 1,59a
Bioactive mulch, Td50 + SF12 5,42a 2,76a 5,18ab 2,54a 3,74a 1,87a 3,22a 1,64a
a- Value followed by the same letter do not dif fer significantly at the P < 0.05 level.
It is already recognized the relationship between AM fungi , GRSP and soil aggregat e
water stability [30, 43] . This relationship is curvilinear over a large range of water stabili ties
[44]. This means that beyond a certain “saturation” GRSP concentration in a given soil,
additional deposition of GRSP will not result in detectable increases in soil aggregate water
stability. However, in many intensely managed agroecosystems, this may not be a major issue,
as water stability it is rather low [30].
The bioactive mulch approach proposed in order to counter act the negative side effects
of cover crop mulches do not reduce the AMF activity and GRSP production into soil. These
prelim inary results shown that the applicati on of the microorganisms (phytopathogens antagonist
like T. viride Td50 or B. amyloliquefaciens B165, diazotroph PGPR like Azospirillum ) increase
water stable aggregates and glomalin (total and easily extractable) on soil, especially in the
shallowest (0 –5 cm) layer of soil. Further studies are necessary at phenomenological level for the
confirmation of responsiveness of GRSP pool in the field to bioactive mulch.

Conclusion s
The treatment with microorganisms antagonist to soil born phytopathogens ( T. vi ride
Td50 and B. amyloliquefaciens B165) did not affect the mean weight diameter of water stable
aggregates. The use of these microorganism into b ioactive mulch agricultural system increase
water stable aggregates ( especially on 0 –5 cm layer of soil) co mpared with cover crop mulch
alone.
Hairy vetch as a cover crop, fungal antagonist ( Trichoderma viride Td50) applied to
mulched hairy vetch and sunflower plant developed from seed inoculated with Azospirillum
brasilense SF12, slightly increase the AMF pr opagule numbers. Bacterial antagonist ( B.
amyloliquefaciens B165) used on the bioactive mulch system, as treatment of cover crop mulch,
combined with sunflower plant developed from seed inoculated with Azospirillum brasilense
SF12, have a more significan t effect on increase of AMF propagule on soil. The abundance of
total intact free AMF spores (SA) and small spored Glomus (SS) species in the field
experimental plot is rather medium and t he differences among total numbers of AMF spores in

the tilled and no-tilled and mulched soil were mostly due to spores in the smallest size class
(under 100 μm).
The field experiment shown that bioactive mulch agricultural system increase glomalin –
related soil protein (GRSP, Bradford reactive soil protein, total and ea sily extractable) on soil,
especially in the shallowest (0 –5 cm) layer of soil. The increase in concentrations on mulched
treatments (and especially on biomulched one) reflect an increase in AMF biomass / AMF
activity. The easily extractable and total glo malin contents were quite low, but similar to those
reported in other studies carried out in soils containing calcium carbonate under similar climatic
conditions .

References
1. Archer, D.W., Reicosky, D.C., 2009, Economic performance of alternative tillage systems in the northern corn
belt, Agron. J. 101: 296-304
2. Arshad, M.A., Lowery, B., Grossman, B., 1996, Physical Tests for Monitoring Soil Quality, in: Doran J.W.,
Jones A. J., eds. Methods for assessing soil quality . Madison, WI. p 123 -141.
3. Bailey, K.L., Lazarovits G., 2003, Suppressing soil -borne diseases with residue management and organic
amendments, Soil & Tillage Research 72: 169–180.
4. Blanco -Canqui, H., Lal, R., 2009, Crop Residue Removal Impacts on Soil Productivity and Environmental
Quality, CRC Crit Rev Plant Sci 28: 139-163
5. Borie, F., Rubio, F., Rouanet, R., Morales, R.L., Borie, A., Rojas C., 2006, Effects of tillage systems on soil
characteristics, glomalin and mycorrhizal propagules in a Chilean Ultisol, Soil Till Res, 88, 253 –261
6. Borie, F., Rubio, R., Morales, A., 2008, Arbuscular mycorrhizal fungi and soil aggregation Rev Ci Suelo Nutr
Vegetal , 8: 9-18.
7. Brimner, T. Boland G.J., 2003, A review of the non -target effects of fungi used to biologically control plant
diseases. Agric Ecos yst Environ 100: 3-16
8. Dill-Macky, R., Jones, R. K., 2000, The effect of previous crop residues and tillage on Fusarium head blight of
wheat, Plant Dis. 84:71-76.
9. Driver, J.D., Holben, W.E., Rillig, M.C., 2005, Characterization of glomalin as a hyphal wall component of
arbuscular mycorrhizal fungi, Soil Biol Biochem 37: 101 –106.
10. Drury, C.F., Tan, C.S. Welacky, T.W., Oloya, T.O., Hamill, A.S. Weaver, S.E., 1999, Red clover and tillage
influence on soil temperature, water content, and corn emergence, Agron. J. 91:101–108
11. Dwivedi, D., Johri, B.N., Ineichen, K., Wray, V., Wiemken, A., 2009 , Impact of antifungals producing
rhizobacteria on the performance of Vigna radiata in the presence of arbuscular mycorrhizal fungi, Mycorrhiza ,
19:559-570.
12. Fageria, N.K., Bal igar, V.C., Bailey B.A., 2005, Role of cover crops in improving soil and row crop
productivity, Comm. Soil Sci Plant Analysis , 19-20: 2733 -2757
13. Franzluebbers, A. J., 2010, Achieving Soil Organic Carbon Sequestration with Conservation Agricultural
Systems in the Southeastern United States, Soil Sci. Soc. Am. J. 74: 347 -357
14. Galvez, L., Douds, D. D. Wagoner, P., Longnecker, L.E, Drinkwater, L.E, Janke, R.R., 1995 , An
overwintering cover crop increases inoculum of VAM fungi in agricultural soil, American J Al ter Agric 10:152–156
15. Green, H., Larsen, J., Olsson, P.A., Jensen D.F, Jakobsen, I., 1999, Suppression of the biocontrol agent
Trichoderma harzianum by mycelium of the arbuscular mycorrhizal fungus Glomus intraradices . Appl. Environ.
Microbiol. 65: 1428 -1434.
16. Johnson , N.C. , O'Dell , T.E., Bledsoe , C.S., 1999 , Standard soil methods for long -term ecological research , in
Robertson , G.P. Bledsoe , C.S, Coleman D.G., Sollins, P. eds., Methods for Ecological Studies of Mycorrhizae ,
Oxford University Press , New Y ork, NY, p. 378–411.
17. Kasper, T.C., Erbach D.C., Cruse R.M.,1990, Corn response to seed -row residue removal, Soil Sci. Soc. Am. J.
54:1112 –17
18. Kemper W.D., Rosenau R.C., 1986, Aggregate Stability and Size Distribution, in: Klute A, ed. Methods of soil
analys is. Part 1. Physical and mineralogical methods . Madison, WI. p 425 -442.
19. Korb, J.C., Johnson, N.C., Covington, W.W., 2003, Arbuscular mycorrhizal propagule densities respond
rapidly to ponderosa pine restoration treatments, J Appl Ecol , 40, 101 -110,
20. Koske , R. E., Tessier, B., 1983. A convenient permanent slide mounting medium, Mycol. Soc. Am. Newsl.
34:59.
21. Kuo, S., Sainju, U.M. Jellum. Jellum, 1997, Winter cover crop effects on soil organic carbon and carbohydrate
in soil, Soil Sci Soc Am J 61:145–152

22. Liu, A., Ma, B. L., Bomke, A. A., 2005, Effects of cover crops on soil aggregate stability, total organic carbon,
and polysaccharides, Soil Sci Soc Am J, 69: 2041 -2048
23. Marquez, C. O., Garcia, V. J., Cambardella, C. A., Schultz, R. C., Isenhart, T. M., 2004, Aggregate -Size
Stability Distribution and Soil Stability, Soil Sci Soc Am J 68: 725 -735.
24. Martínez A., Obertello M., Pardo A., Ocampo, J.A., Godeas A., 2004, Interactions between Trichoderma
pseudokoningii strains and the arbuscular mycorrhizal fungi Glomu s mosseae and Gigaspora rosea , Mycorrhiza 14:
79-84.
25. Martínez -Medina, A., Pascual, J. A., Pérez -Alfocea, F., Albacete, A., Roldán, A., 2010, Trichoderma
harzianum and Glomus intraradices modify the hormone disruption induced by Fusarium oxysporum infection in
melon plants, Phytopathology 100:682-688.
26. Nichols, K.A., Wright, S.F., 2005. Comparison of glomalin and humic acid in eight native U.S. soils, Soil Sci
170, 985 –997.
27. Oancea, F., Dinu, S., Constantinescu, F., Sicuia, O., Zamfiropol, R., 2010, Procedeu d e cultivare a plantelor în
mulci bioactiv format din culturi de protecție de leguminoase, OSIM patent application A217/2010.
28. Oehl F., Sieverding, E., Ineichen, K., Mäder, P., Boller, T., Wiemken, A., 2003 , Impact of land use intensity on
the species diver sity of arbuscular mycorrhizal fungi in agroecosystems of Central Europe . Appl Environ Microbiol
69: 2816 –2824
29. Pereyra, S. A., Dill -Macky, R., 2008., Colonization of the residues of diverse plant species by Gibberella zeae
and their contribution to Fusariu m head blight inoculum. Plant Dis . 92:800-807
30. Rillig, M.C., Wright, S.F., Eviner, V.T., 2002, The role of arbuscular mycorrhizal fungi and glomalin in soil
aggregation: comparing effects of five plant species, Plant Soil 238:325–333.
31. Sainju, U. M., Whitehe ad, W. F., Singh, B. P., 2003, Cover crops and nitrogen fertilization effects on soil
aggregation and carbon and nitrogen pools, Can J Soil Sci 83:155–165.
32. Schenck, N. C., Perez, Y., 1990, Manual for the identification of VA mycorrhizal fungi , 3rd ed. Sy nergistic
Publications, Gainesville, Fla.
33. Stanil ă, S. 2006, Influența tehnologiilor de conservare a solului asupra eroziunii hidrice, Agricultura – Stiință
si practică , 59-60: 61-67.
34. Stockdale, E.A., Watson, C.A., 2009, Biological indicators of soil quality in organic farming systems, Ren
Agric Food Sy s 24: 308 –318
35. Triplett, G.B., Jr., Dick W.A., 2008, No -till crop production: A revolution in agriculture! Agron. J. 100:S153 –
S165.
36. Vargas Gil, S., Meriles, J., Conforto, C., Figoni, G., Basanta, M., Lovera, G., March, G. J., 2009, Field
assessment of soi l biological and chemical quality in response to crop management practices, World J Microbiol
Biotech 25:439-448.
37. Vázquez, M.M., Cesar, S., Azcon, R., Barea, J.M., 2000, Interactions among arbuscular mycorrhizal fungi and
other microbial inoculants ( Azosp irillum , Pseudomonas , Trichoderma ) and their effects on microbial population and
enzyme activities in the rhizosphere of maize plants. Appl Soil Ecol , 15:261–272.
38. Vetsch, J.A., Randall, G.W., 2000, Enhancing no -tillage systems for corn with starter fertili zers, row cleaners,
and nitrogen placement methods, Agron. J. 92: 309-315.
39. Villamil, M. B., Bollero, G. A., Darmody, R. G., Simmons, F. W., Bullock, D. G., 1995, No-Till Corn/Soybean
Systems Including Winter Cover Crops: Effects on Soil Properties, Soil Sc i. Soc. Am. J. 70: 1936 -1944.
40. Wilson, G.W.T. , Rice, C.W. , Rillig, M.C. , Springer, A., Hertnett, D.C., 2009 , Soil aggregation and carbon
sequestration are tightly correlated with the abundance of arbuscular mycorrhial fungi: Results fom long -term field
expe riments , Ecol Lett 12: 452–461
41. Wright, S.E., Nichols, K.A., Schmidt, W.F. 2006. Comparison of efficacy of three extractants to solubilize
glomalin on hyphae and in soil. Chemosphere . 64:1219 -1224.
42. Wright, S.F., Franke -Snyder, M., Morton, J.B., Upadhyaya, A ., 1996, Time -course study and partial
characterization of a protein on hyphae of arbuscular mycorrhizal fungi during active colonization of roots, Plant
Soil 181:193–203.
43. Wright, S.F., Starr, J.L., Paltineanu, I.C., 1999. Changes in aggregate stability a nd concentration of glomalin
during tillage management transition. Soil Sci Soc Am J 63: 1825 –1829.
44. Wright, S.F., Upadhyaya, A., 1996, Extraction of an abundant and unusual protein from soil and comparison
with hyphal protein of arbuscular mycorrhizal fun gi, Soil Sci 161, 575 –586.
45. Wright, S.F., Upadhyaya, A., 1998. A survey of soils for aggregate stability and glomalin, a glycoprotein
produced by hyphae of arbuscular mycorrhizal fungi, Plant Soil 198, 97–107.
46. Wuest, S.W., 2007, Surface versus incorporated residue effects on water -stable aggregates Soil Till Res
96:124–130
47. Xiao, X., Chen, H., Chen, H., Wang, J., Ren, C., Wu L.J., 2008, Impact of Bacillus subtilis JA, a biocontrol
strain of fungal plant pathogens, on arbuscular mycorrhiza formation in Zea may s, World J Microbiol Biotech , 24,
133-1137.

Efectele sistemului de agricultură conservativă cu mulci bioactiv asupra agregatelor stabile în apă, a
propagulelor micorizale și a glomalinei dintr -un cernoziom cambic.

(Rezumat)

Am dezvoltat un sistem de agricultură conservativă pe bază de "mulci bioactiv", format
dintr -o cultură de protecție în timpul iernii , resturile ve getale rezultate din cultura de protecție
fiind inoculate cu microorganisme antagoniste fitopatogenilor proveniți din sol, iar semințele
culturii principale fiind inoculate cu bacterii diazotrofe și cu acțiune de favorizare a creșterii
plantelor.
Am evaluat efectele acestui sistem de agricultură alternativă asupra agregatelor stabile în
apă (WSA) , propagulelor de ciuperci arbusculo -micoriz ale și glomalinei (ușor -extractibilă și
totală ). Experimentul nostru de câmp a fost realizat pe un cernoziom cambic (Amzacea,
Dobrogea), folosind pe post de cultură de protecție măzărichea păroasă și floarea -soarelui cu rol
de cultură valorificabilă comerc ial. Experimentul a relevat faptul că sistemul cu mulci bioactiv
favorizează creșterea agregatelor stabile în apă și a glomalinei (totale și ușor-extractibile), în
special în stratul de la suprafața solului (0 -5 cm). Măzărichea păroasă utilizată cu rol de cultură
de protecție, ciuperca microscopică antagonist ă (Trichoderma viride Td50) aplică pe măzărichea
mulcită și plantele de floarea -soarelui dezvoltate din semințe inoculate cu Azospirillum
brasilense SF12, crește ușor numărul de propagule AMF. Antagonis tul bacterian ( B.
amyloliquefaciens B165) folosit în sistemul de mulci bioactiv ca tratament al mulciului format
din cultura de protecție, în combinație cu plante de floarea -soarelui dezvoltate din semințe tratate
cu Azospirillum brasilense SF12, are un ef ect mai semnificativ de creștere a numărului de
propagule AMF din sol.
Rezultatele demonstrează faptul că sistemul de agricultură conservativ pe bază de mulci
bioactiv crește activitatea ciupercilor de endomicoriză pe cernoziomul cambic din zona
Dobrogei.

Aknowledgement : The research work was supported by grant 135127/135194, 17.07.2009 –
30.06.2011 , SCG UMP MAKIS MADR -World Bank, „ Sistem de agricultură alternativă pe bază
de mulci vegetal bioactivat format din culturi verzi ”.