INFLUENCE OF FERTILIZATION ON SOIL MICROFLORA

Cercetări Agronomice în Vol. XLII , No. 2 (138) / 2009

INVESTIGATIONS ON THE INFLUENCE OF FERTILIZATION AND OF ONOBRYCHIS VICIIFOLIA SCOP. AND BROMUS INERMIS LEISS. MIXTURE ON SOIL MICROFLORA

E. ULEA*, F.D. LIPŞA, Nicoleta IRIMIA, Gabriela Mihaela BALAN

University of Agricultural Sciences and Veterinary Medicine, Iaşi

Received September 18, 2008

ABSTRACT - This paper presents the results of an experiment conducted on two Keywords: fertilization, microbial activity, temporary meadows, situated in the soil, temporary meadow Southern area of the , set up by sowing a mixture made up of Onobrychis REZUMAT - Cercetări privind influenţa viciifolia Scop. (sainfoin) and Bromus fertilizării şi a amestecului de Onobrychis inermis Leiss. (smooth bromegrass) at viciifolia Scop. şi Bromus inermis Leiss. different rates. We have analysed the soil asupra microflorei solului. Lucrarea microbiological activity of the two prezintă rezultatele obţinute în cadrul unor temporary meadows, as influenced by the experienţe, amplasate în două pajişti fertilization with an unconventional product temporare din zona sudică a Câmpiei (Vinasse Rompak - 3% N, 0.5% P, 7% K Moldovei, înfiinţate prin însămânţarea unui and pH 7-8) and with different rates of amestec de Onobrychis viciifolia Scop. organic and chemical fertilizers. The (sparcetă) şi Bromus inermis Leiss. (obsigă objective of this investigation was to isolate nearistată), în proporţii diferite. S-a urmărit and quantify the microbial population found activitatea microbiologică a solului din cele in soil (Gram-positive bacteria, Gram- două pajişti temporare, sub influenţa negative bacteria, micromycetes and fertilizării cu un produs neconvenţional nitrogen-fixing bacteria), thus establishing (Vinassa Rompak - 3% N, 0.5% P, 7% K, their percent, main fungus genera, which pH 7-8), cu îngrăşăminte organice şi activate in soil and their activity level for chimice. S-au determinat grupele principale each variant. The results have shown the de microorganisme prezente în sol (bacterii influence of the fertilization on the total Gram pozitive, bacterii Gram negative, number of microorganisms, on the micromicete şi fixatori de azot), stabilindu- relationships between the main groups se procentul de participare al acestora, (bacteria and fungi) and on the various principalele genuri de ciuperci care micromycete species, determined in each activează şi nivelul activităţii acestora variant of our experiment. pentru fiecare variantă. Rezultatele au

* E-mail: [email protected]

47 E. ULEA ET AL. evidenţiat o activitate microbiană diferită şi rates and combinations. This is un spectru divers de micromicete între toate necessary for soil balance as concerns variantele avute sub observaţie, ca urmare a the natural microflora from soils, fertilizării diferenţiate şi a raportului diferit între cele două specii. which maintains soil health and, implicitly, a constant productive Cuvinte cheie: activitate microbiană, potential. fertilizare, sol, pajişte temporară The goal of this study was to analyse and compare the INTRODUCTION microbiological activity from soil, occupied with two types of temporary Maintaining or improving soil meadows, as influenced by the organic matter is an important fertilization with an unconventional objective for each sustainable product (Vinasse Rompak) and with agriculture system. The organic organic and chemical fertilizers. matter loss has a negative effect on soil chemical, physical and biological MATERIALS AND METHODS characteristics. The diminution in soil microbial activity is alarming, The trial was conducted on a 2-3% because of the active role of slope field from the Ezăreni Farm, which microorganisms from soil: decay of belongs to the University of Agricultural biological residues, mineralization of Sciences and Veterinary Medicine, Iaşi. the organic matter, formation and Soil is a clayey loam cambic chernozem, stabilization of soil aggregates, etc. weakly degraded, with pH comprised between 6.7 and 6.8, humus content 2.73- (Cardon et al., 2007; Papacostea, 2.93%, 51-55 ppm P O , 314-336 ppm 1976). 2 5 K2O and 184-187 ppm CaO. The area is Under natural ecosystems, the characterized by mean annual losses of main mineral elements from temperatures of 9.6o C, annual rainfall of soil, necessary to plant nutrition, are 517.8 mm and air relative humidity of compensated by fixing and 69%. solubilisation processes, while in the From the physical-geographical systems used in agriculture, these viewpoint, this territory is found in the losses exceed the rate of replacement. Southern area of the Moldavian Plain, This leads to a lack of balance which is named the Lower Jijia Plain and the Plain, being situated in the between the needs of nutritive South-Western extremity of this natural elements and the opportunity of zone. covering these needs (Saratchandra et The studied factors were: al., 1988; Varma et al., 2004). Factor A = mixture of perennial This problem may be solved by legumes and grasses: A1 = Onobrychis using conventional (organic and viciifolia 20% + Bromus inermis 80%; A2 mineral fertilizers) or unconventional = Onobrychis viciifolia 70% + Bromus products (Vinasse), on the condition inermis 30%. of their application in well-determined Factor B = fertilization: B1 = unfertilized control; B2 = N200P100 kg/ha;

48 INFLUENCE OF FERTILIZATION ON SOIL MICROFLORA

B3 = 30 t manure /ha and B4 = 5 t/ha genera were emphasized by using the Vinasse. Ashby medium (Constantinescu, 1974). For determining the number of Sowing was done by introducing a microorganisms per 1 g soil, we have ml of dilution in each Petri dish with used the culture method in Petri dishes. melted and cooled medium at 45°C. The Soil samples were gathered in paper bags, sown dishes were incubated in a by means of a metallic spatula and the thermostat at 28°C. The number of used material was previously sterilized. bacterial colonies was determined at 24 Soil was sampled at 10 cm depth and then hours and the fungus colonies at 5 days; samples were processed by grinding and counting was done by naked eye, using a homogenization in a sterile mortar. Soil marker. At high densities, the Wolfhügel dilutions were prepared according to the plate was used (Larpent et al., 1990). method of successive dilutions and sowing was done in Petri dishes, by the RESULTS AND DISCUSSION incorporation in medium. For an easy identification of The analysis of the obtained data colonies, we have used different culture on the two types of temporary mediums, specific to systematic groups. meadows has shown a significant Thus, for determining the total number of microorganisms, we have used the simple increase of soil biological activity in PDA (potato-dextrose-agar) medium, for all the fertilized variants, compared determining the number of Gram-positive with the unfertilized control, except bacteria, we have used the PDA with the variant sown with a mixture made streptomycin (35 ppm) medium and for up of Onobrychis viciifolia 20% + determining the number of micromycetes, Bromus inermis 80%, fertilized with we have used the PDA with Bengal Pink Vinasse, where the number of (33 ppm) medium. Nitrogen fixing- microorganisms per soil gram was bacteria of Azotobacter and Clostridium equal to their number from the control (Table 1).

Table 1 – Biological activity of soil occupied with temporary meadows

Total Bacteria Fungi N Fertilization 2 Mixture micro- Fixing variant thousands/ thousands/ organisms % % bacteria g soil g soil A1 Unfertilized 277 157 56.7120 43.3 x A2 control 444 250 56.3 194 43.7 x A1 1103 1033 93.770 6.3 x N200P100 kg/ha A2 940 810 86.2 130 13.8 x A1 30 t/ha 414 314 75.8100 24.2 x A2 manure 885 785 88.7 100 11.3 x A 270 210 77.860 22.2 x 1 5 t/ha Vinasse A2 599 549 91.7 50 8.3 x A1 = Onobrychis viciifolia 20% + Bromus inermis 80%; A2 = Onobrychis viciifolia 70% + Bromus inermis 30%; x - The presence of nitrogen-fixing bacteria.

49 E. ULEA ET AL.

In all the studied variants, we inermis 80% (A1), the highest activity isolated nitrogen-fixing species was recorded in B2 Variant (N200P100 belonging to Azotobacter and kg/ha), followed by B3 Variant (30 Clostridium genera. t/ha manure). In the mixture made up In both mixtures used for sowing of Onobrychis viciifolia 70% + the two types of temporary meadows, Bromus inermis 30% (A2), the same the highest biological activity was fertilization variant has signalled recorded in the variants fertilized with the highest number of nitrogen and phosphorus-based microorganisms/g soil, followed by fertilizers. In the mixture made up of B3 (30 t/ha manure N150) and B4 (5 Onobrychis viciifolia 20% + Bromus t/ha Vinasse) (Figure 1).

Fig. 1 – Differentiation of the microbiological activity per fertilization variants and type of temporary meadow

Analysing the ratio between the (B1) and 91.7% (B3), in case of A2 main groups of microorganisms found mixture (Figure 2). The change of the in the soil occupied by the two types ratio between the main systematic of temporary meadows, we found groups, in order to favour the significant differences among all the numerical increase in bacteria or fertilization variants, except the fungus species to the prejudice of the unfertilized control, where the ratio other microorganism group may be between bacteria and micromycetes explained by the application of was identical for the two types of fertilizers or other unconventional meadows. The best represented products (Vinasse Rompak). The high microorganism group for all the rate of soil bacteria in all the fertilized fertilization variants and for each type variants and for both types of of meadow is that of bacteria that are meadows may be explained by their between 56.7% (B1) and 93.7% (B2) competition against micromycetes and of the total microorganisms, in case plants as concerns some nutrients, and of A1 mixture, and between 56.3% by increasing the soil concentration in

50 INFLUENCE OF FERTILIZATION ON SOIL MICROFLORA nutrients, due to fertilizers (especially, increase in the bacterial populations nitrogen and phosphorus fertilizers), (Ulea et al., 1999; Wood, 1989). which determined an exponential

Fig. 2 – Main groups of microorganisms for each fertilization variant and type of temporary meadow

The investigations conducted on variants, compared to the unfertilized the frequency of micromycete genera control, at both used mixtures have shown a diminution in the (Bontea, 1986; Gilman, 1959) (Table number of genera, in the fertilized 2).

Table 2 – Isolated micromycete genera found in the soil occupied by temporary meadows

Fertilization Mixture Isolated micromycete genera variant Penicillium, Aspergillus, Fusarium, Alternaria, Rhizopus, A 1 Unfertilized Mycogone control Penicillium, Aspergillus, Fusarium, Alternaria, Robillarda, A 2 Cladosporium, Trichoderma, Mucor, Rhizopus A1 Penicillium, Aspergillus, Alternaria, Rhizopus N200P100 kg/ha Penicillium, Fusarium, Alternaria, Robillarda, A 2 Cladosporium, Trichoderma, Rhizopus A Penicillium, Fusarium, Aspergillus, Rhizopus, Mycogone 1 30 t/ha Penicillium, Aspergillus, Fusarium, Melanospora, A manure 2 Cladosporium, Trichoderma, Mucor, Rhizopus A1 Penicillium, Trichoderma, Rhizopus 5 t/ha Vinasse Penicillium, Aspergillus, Fusarium, Trichoderma, A 2 Rhizopus A1 = Onobrychis viciifolia 20% + Bromus inermis 80%; A2 = Onobrychis viciifolia 70% + Bromus inermis 30%;

51 E. ULEA ET AL.

Fig. 3 – Micromycete species found in each fertilization variant (B1, B2, B3 and B4) and type of temporary meadow (A1, A2)

We have also noticed that the viciifolia 70% + Bromus inermis 30% number of isolated fungus genera, in (A2), we identified species belonging the meadow sown with A1 mixture to 10 genera (Penicillium, was more reduced than in case of A2 Aspergillus, Fusarium, Alternaria, mixture. In the mixture made up of Robillarda, Cladosporium, Onobrychis viciifolia 20% + Bromus Trichoderma, Melanospora, Mucor inermis 80% (A1), we have isolated and Rhizopus). This difference may be species belonging to six micromycete explained by the selection of some genera (Penicillium, Aspergillus, specific fungus genera, according to Fusarium, Alternaria, Rhizopus, the biochemical features of root Mycogone and Trichoderma), while in secretions in the two sown mixtures the mixture made up of Onobrychis (Yevdokimov et al., 2008; Zarnea,

52 INFLUENCE OF FERTILIZATION ON SOIL MICROFLORA

1994). Thus, the species belonging to mixture and respectively, between Robillarda, Cladosporium, 35.7 and 60%, for the A2 mixture. Melanospora and Mucor genera were The micromycete species from isolated only from the temporary the A1 mixture were completed by meadow, sown with the mixture made Fusarium, Aspergillus and Rhizopus up of Onobrychis viciifolia 70% + genera, and from the A2 mixture, by Bromus inermis 30%, while Trichoderma, Fusarium, Mycogone genus was identified only Cladosporium and Rhizopus genera in the mixture made up of Onobrychis (Figure 3) viciifolia 20% + Bromus inermis 80% The analysis on the number of (Figure 3). micromycete colonies/Petri dish has Among the determined shown that among all the fertilization micromycetes in all the studied variants, the lowest number of variants, we pointed out Penicillium colonies was recorded in the variant genus, which was isolated at a rate fertilized with Vinasse 5 t/ha (B4), comprised between 50 and 69.9% of and the highest number, in the the total identified genera for the A1 unfertilized control (B1) (Figure 4)

Fig. 4 – Determination of the number of micromycete colonies/ Petri dish, for each fertilization variant and type of temporary meadow

CONCLUSIONS Between the two mixtures used for setting up the meadows, the Our observations have shown a highest microbial activity in B1 very good activity of aerobe and (unfertilized), B3 (30 t/ha manure) anaerobe nitrogen-fixing bacteria that and B4 (Vinasse 5 t/ha) was recorded live free in soil and complete the at the mixture made up of Onobrychis activity of bacteria found in the viciifolia 70% + Bromus inermis 30% nodosities of legumes. (A2), and in B2 (N200P100 kg/ha) at the

53 E. ULEA ET AL. mixture made up of Onobrychis Gilman J., 1959 – A Manual of Soil Fungi, viciifolia 20% + Bromus inermis 80% the Iowa University Press, Iowa, USA (A1). Hiltner L., 1904 - Über neuere The microbial activity has shown Erfahrungen und Probleme auf dem a significant increase at both mixtures Gebiet der Bodenbakteriologie und used for sowing, in all the fertilized unter besonderer Berücksichtigung variants, compared to the unfertilized der Gründüngung und Brache, Arb. Dtsch. Landwirt. Ges. 98, p. 59-78 control. Larpent J.P., Larpent-Gourgand M., At both mixtures made up of 1990 - Mémento technique de Onobrychis viciifolia 20% + Bromus Microbiologie, Lavoisier, Paris inermis 80% (A1) and Onobrychis Papacostea P., 1976 - Biologia solului (Soil biology), Edit. Ştiinţifică şi viciifolia 70% + Bromus inermis 30% Enciclopedică, Bucureşti (A2), the highest number of Sarathchandra S.U., Perrott K.W., microorganisms was recorded in the Boase M.R., Waller J.E., 1988 - Seasonal changes and the effects of variant fertilized with N200P100 kg/ha. In both trials, we found a fertilizer on some chemical, biochemical and microbiological diminution in the number of identified characteristics of high-producing fungus genera at the fertilized pastoral soil, Biology and Fertility of variants, compared with the Soils 6, p. 328-335 unfertilized control. Ulea E., Samuil C., 1999 - Dinamica activităţii biologice a pajiştilor In all the studied variants, from temporare situate pe teren erodat all the isolated micromycete species, (Dynamics of the biological activity Penicillium genus had the highest of temporary meadows situated on frequency; it was followed by eroded land), Lucr. şt., seria Fusarium, Aspergillus, Trichoderma Agronomie, vol. 42, U.Ş.A.M.V. Iaşi, p. 90-96 and Rhizopus genera. Varma S., Abbott L., Werner D., Hampp R., 2004 - Plant surface REFERENCES microbiology, Springer Verlag Berlin, Germany Wood M., 1989 - Soil Biology, Blackie and Bontea Vera, 1986 - Ciuperci parazite şi Son et al, London a Saint-Amand.C saprofite din România (Parasite and Yevdokimov I., Gattinger A., Buegger saprophyte fungi from ), F.,Munch J.C., Schloter M., 2008 - Edit. Acad. R.S.R. Bucureşti Changes in microbial community Constantinescu Ovidiu, 1974 - Metode şi structure in soil as a result of tehnici în micologie (Methods and different amounts of nitrogen techniques in mycology), Edit. fertilization, Biol Fertil Soils 44, p. Ceres, Bucureşti 1103–1106 Cardon G. Zoe, Whitbeck L. Julie, 2007 Zarnea G., 1994 - Tratat de Microbiologie - The Rhizosphere: An Ecological generală (Treatise of general Perspective, Elsevier Academic microbiology), Edit. Academiei Press, USA Române, Bucureşti, vol. V Eliade G., Ghinea L., Ştefanic G., 1975 -

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