0003-6838/01/3703- $25.00 © 2001

MAIK “Nauka

/Interperiodica”0322

Applied Biochemistry and Microbiology, Vol. 37, No. 3, 2001, pp. 322–326. Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 37, No. 3, 2001, pp. 374–378.Original Russian Text Copyright © 2001 by Lisina, Garan’kina, Kruglov.

Extensive application of plant protective chemicalsfor agricultural needs results in soil pollution with pes-ticides and their derivatives, thus creating environmen-tal and sanitary-hygienic problems.

Microbes are the major factors of polluted soil self-remediation. A substantial number of bacterial, fungal,and actinomycete genera capable of degrading and con-verting these substances was isolated from soils andother environments [1–3].

Several attempts were made to use microorganismsas destructors in pesticides and other hazardous sub-stances detoxification in soils [4–8]. A high initial inoc-ulation level (10

6

to 10

7

cells per 1 g soil) was noted asa necessary condition for a substantial effect to beachieved [2, 8].

Microbial removal of organic poisons from soils is amild and environmentally friendly procedure. How-ever, the question arises as to the effect of high loadingof microbial destructors on native soil microflora andplants.

We have isolated and studied microbial strains capa-ble of transforming and degrading a number of organo-phosphorus compounds used as insectoacaricides [9–13].They substantially promoted degradation of such acar-icides as gardona, aktelli, phosphamide, and carbophosin inoculated soils.

The aim of this work was to study the effect of inoc-ulation of peach substrates by microbial destructors ofpesticides on the growth and development of a numberof greenhouse-cultivated vegetables.

MATERIALS AND METHODS

Spore-forming bacteria

Bacillus megaterium

501isolated from common loamy black soil (Kokchetavoblast, Kazakhstan) that degrades several organophos-

phorus compounds, including insecticides gardona,aktelli, rogor, carbophos [9, 11, 12], was deposited inthe culture collection of the All-Russia Institute ofAgricultural Microbiology.

Black yeast

Exophialia nigrum

st. A-29 obtainedfrom the collection of the genetic laboratory of theInstitute of Biology, the State University of Irkutsk,readily degraded gardona insecticide [13].

Microorganisms were cultivated in Erlenmeyer flaskson a rotary shaker under extensive aeration.

B. megate-rium

was grown on a medium containing 0.05% of(NH

4

)

2

SO

4

, 0.03% MgSO

4

· 7H

2

O, 0.03% NaCl,0.03% CaCO

3

, 0.45% Na

2

HPO

4

, 0.3% KH

2

PO

4

,1.0% molasses, and 0.03 to 0.05% yeast extract (pH 6.8to 7.2); that of

E. nigrum

contained 0.3% (NH

4

)

2

SO

4

,0.07% MgSO

4

· 7H

2

O, 0.05% NaCl, 0.04% Ca(NO

3

)

2

,0.01% K

2

HPO

4

, 0.1% KH

2

PO

4

, 20 g of molasses, and0.3 g of yeast extract (pH 5.5 to 5.7).

Pilot scale experiments were conducted by means ofsubmerged cultivation of microorganisms in 100-l fer-mentors under extensive aeration at 30

°

C.

In the vegetation and field experiments, liquidmicrobial cultures from the late exponential phase wereused, i.e., 2-d culture of

B. megaterium

and 4-d cultureof

E. nigrum

A-29 (bacterial count 10

8

to 10

9

, yeastcount 10

7

to 10

8

cells per ml, respectively).

Soil or peach substrate was inoculated by liquidmicrobial cultures by means of watering. Inoculateloading (10

5

to 10

7

cells per 1 g substrate depending onthe experiment and microorganism utilized) wasobtained by appropriate water dilution of the liquid cul-tures. In the control experiments, soil was poured withwater or liquid cultures autoclaved at 1 atm for 15 min.

The following substrates were used in the vegetationexperiments.

The Effect of Soil Inoculation with Microbial Pesticide Destructors on Plant Growth and Development

T. O. Lisina, N. G. Garan’kina, and Yu. V. Kruglov

All-Russia Research and Development Institute for Agricultural Microbiology, Russian Academy of Agricultural Sciences,St. Petersburg, 196608, Russia

e-mail: chief@riam.spb.su

Received March 13, 2000

Abstract

—Soil inoculation with liquid cultures of

Bacillus megaterium

501 and

Exophiala nigrum

A-29 capa-ble of degrading several organophosphorus pesticides accelerated the growth and development of experimentalplants, the formation of their generative organs, and improved their productivity. This was particularly observedunder stress plant growth conditions on phytotoxic peach substrates. The microorganisms inoculated can prob-ably degrade the phytotoxins present in soils, thereby favoring plant development.

APPLIED BIOCHEMISTRY AND MICROBIOLOGY

Vol. 37

No. 3

2001

THE EFFECT OF SOIL INOCULATION WITH MICROBIAL PESTICIDE DESTRUCTORS 323

Substrate 1: a 1 : 1 mixture of treated turf-podsoliclight loamy soil and peach-mineral substrate containingsurface peach at a low decomposition degree, lime, andmineral fertilizers (a

micoparnik

produced by the peachplant Pelgora, Leningrad oblast).

Substrate 2: heavily exploited peach soil from theLeto ltd. industrial greenhouse after 5 years of exploi-tation, demonstrating a clear soil exhaustion effect.

Substrate 3: freshly prepared limed surface peachsupplemented with mineral fertilizers obtained fromthe Tsvety industrial greenhouse production and tradecompany (St. Petersburg), where a massive loss of pinkseedlings was observed.

Substrate 4: briquettes of hot-pressured surfacepeach.

The substrates were placed in clay or peach pots andsown by vegetable seeds. In the large-scale experi-ments, peach briquettes were used for tomato (cultivarGrain) seedling cultivation. Two months later, the seed-lings, along with the peach briquettes, were planted

into the peach ground of the industrial greenhouses.Vegetable crop yields were counted during a ripeningperiod of 3 weeks. The total experimental land area wasequal to 840 m

2

.Peking cabbage (

Brassica campestris

L.), cultivarKhibinskaya, celery (

Apium graveolens

L.), cultivarYablochnyi, cucumber (

Cucumis sativus

L.), cultivarMuromskii, tomatoes (

Lycopersium esculentum

L.),cultivars Novinka Pridnestrov’ya, Nevskie, Grain, andpepper (

Capsicum frutescens

L.) were used as test cul-tures. Statistical analysis of the results was performedat a confidence level

P

of 0.95 [14].

RESULTS AND DISCUSSION

The results of vegetation experiments have shownno negative effect of presown soil treatment by liquidcultures of

B. megaterium

501 and

E. nigrum

A-29 onthe development of plant test cultures. Moreover, in thevast majority of experiments, their productivity was sub-stantially enhanced. A particular effect was observed for

Table 1.

The effect of soil (substrate 1) inoculation by microorganisms on the Peking cabbage crop yields

Experimental setMass of 10 plants, g

control set

Bac. megaterium E. nigrum

No fertilizers

56.0

±

0.37 81.0

±

1.35 123.0

±

1.19

Mineral fertilizers (N, P, K)

93.0

±

0.62 149.0

±

1.73 162.0

±

0.91

Table 2.

The effect of soil (substrate 1) inoculation by microorganisms on celery crop yields

Substrate treatment procedure Inoculate loading

, 10

6

per g soil

Mass of ten plants, g

g %

of control plants

Control set (water)

0 31.0

±

2.3 100

Liquid culture

Bac. megaterium

1.0 41.0

±

5.3 132

"

10.0 53.0

±

7.5 171

Liquid culture

E. nigrum

0.1 43.0

±

3.8 139

"

1.0 75.0

±

4.0 242

Table 3.

Effects of liquid microbial cultures on the growth of cucumber plants on peach substrate 1

Substrate treatment procedure

Mass of one plant

after six weeks after nine weeks

g

excess above control

, % g

excess above control

, %

Control set (water)

31 – 49 –

Culture liquid of

Bac. megaterium

43 32 77 57

Culture liquid of

Bac. megaterium

(sterile) 36 16 55 12

Culture liquid of

E. nigrum

63 103 75 53

Culture liquid of

E. nigrum

(sterile) 45 45 64 30

P

0.95

7.2%

P

0.95

8.1%

324

APPLIED BIOCHEMISTRY AND MICROBIOLOGY

Vol. 37

No. 3

2001

LISINA

et al

.

leaf vegetables. For example, crop yields of Pekingcabbage in the presence of

B. megaterium

and

E. nigrum

increased 1.45 to 1.7 and 2 times, respec-tively (Table 1). The positive effects of soil inoculationby the microorganisms studied were observed both inthe presence and in the absence of mineral fertilizers.The same picture was obtained in the set of experi-ments with celery. An increase in inoculation loadingby one decimal order of magnitude resulted in morethan twofold greater crop yields (Table 2).

Table 3 shows the effect of liquid cultures of

B. megaterium

501 and

E. nigrum

A-29 introduced tothe same substrate on cucumber test culture growth anddevelopment. Both microorganisms accelerated theplant growth, which resulted in increases in the stemheight and plant weight. The effects of autoclavedmicrobial cultures were substantially smaller than thoseof vital ones. This enables a conclusion to be made thatthe positive effect of microorganisms on plant growthand development can be attributed, on the one hand, tothe presence of biostimulators in the culture liquidsand, on the other hand, to their active function in soils.The latter was confirmed in the set of experiments withpeach substrates toxic to plants. For example, cropyields of Peking cabbage test plant cultivated on peach-mineral ground from the Tsvety production and tradecompany, which caused a massive loss of pink seed-lings (substrate 3), increased by 1.5 to 2 times aftertreatment with

B. megaterium

, whereas very weakplant development was observed in the control experi-mental set. A similar picture was obtained in the exper-iments with tomatoes (Table 4): 50% of the plants werelost during cultivation of the tomato seedlings in peach

pots filled with the same substrate, whereas the growthof the remaining ones was extremely suppressed. Incontrast, when the substrate-containing pots were pre-treated with liquid microbial cultures, the inhibitoryeffect diminished, and the plants developed as usual.Their biomass weight exceeded that of the control by2.5 to 5 times. An earlier formation of generativeorgans was also observed. Supplementation with com-plex nitrogen, phosphorous, and potassium fertilizersdid not substantially affect the growth and developmentof the tomato seedlings, thus indicating the sufficiencyof mineral nutrition constituents.

Hot-pressured peach briquettes spoiled because ofhigh phytotoxicity (substrate 4) also provided unfavor-able conditions for plant development. The growth ofsweet pepper seedlings on this substrate was sup-pressed; the thick stems were covered by small leaveswith clear signs of chlorosis. Presown treatment ofpeach briquettes with

B. megaterium

501 or

E. nigrum

resulted in a fivefold or threefold increase, respectively,in plant biomass. The stem height increased twofold ascompared to the control set. This also lead to a moreextensive formation of plant generative organs (Table 5).

Similar results were obtained in the large-scaleexperiments, where tomato seedlings were grown onthe same peach briquettes. Their pretreatment with liq-uid microbial cultures resulted in the formation of well-developed tomato seedlings, whose replantation in thegreenhouse ground substantially accelerated tomatoripening and increased crop yields, particularly at theearly stages of plant development (Table 6).

Having been spoiled because of high phytotoxicity(soil exhaustion phenomenon) after extensive exploita-

Table 4.

Effects of microorganisms on the growth of tomatoes (cultivar “Nevskii”) in peach pots

*

Soil treatment procedureThe number of plants

Plant mass, goriginal lost blossomed

Control set (water)

8 4 0 1.4

Mineral fertilizers (N, P, K)

8 3 0 1.1

Liquid culture of

Bac. megaterium

8 0 8 7.0

Liquid culture of

E. nigrum

8 2 4 3.5

P0.95 9.7%

* Peach substrate from the Tsvety production and trade company (substrate 3).

Table 5. The growth of pepper on pressurized peach briquettes inoculated by microorganisms

Substrate treatment procedure Plant height, mm Mass of ten plants, g Flowers and embryo per ten plants

Control set (water) 101 56 35

Liquid culture of E. nigrum 224 257 87

Control set (water) 90 46 15

Liquid culture of Bac. megaterium 191 140 85

P0.95 % 7.8 8.5 5.9

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 37 No. 3 2001

THE EFFECT OF SOIL INOCULATION WITH MICROBIAL PESTICIDE DESTRUCTORS 325

tion for many years, the industrial grounds obtainedfrom greenhouses of Leto Ltd. (St. Petersburg) were ofour particular interest.

Table 7 summarizes the results of a set of vegetationexperiments with these grounds and sweet pepper orcucumber as plant test cultures. In the control set, theplants were weakly developed and their root systemwas atrophied. However, there was no sign of infectiousdiseases. Inoculation of the ground with the microor-ganisms studied diminished the inhibitory effect of thesoil substrate on plants. An extensive development ofthe root system was observed whose mass 7 to 14-foldexceeded that of the control. The mass of the green partof the plant increased threefold to fivefold. This enablesa conclusion to be made that the positive effect of soilinoculation by the studied microorganisms was morepronounced in extreme conditions, when, for severalreasons, the soil substrate became phytotoxic to culti-vated plants.

Phytotoxicity of the peach grounds used in our setsof experiments can be attributed to several factors, e.g.the allelopathic effect of peach-forming plants [15, 16];the production and accumulation of organic mattertransformation products during anaerobic peach forma-tion [17, 18]; humus hydrolysis during peach self-heat-ing [17]; and the soil exhaustion effect after extendedexploitation of organogenic grounds [16, 19, 20]. Inany event, this can be attributed to the formation andaccumulation of toxicants of agricultural plants. It ispossible to conclude that soil inoculation with B. mega-terium 501 and E. nigrum A-29 results in decomposi-tion of poisoning substances and detoxification of thesoil substrate, which resulted in a positive effect of

these microorganisms on plant growth and develop-ment.

Hence, introduction of the microorganisms studiedinto soil not only accelerated the degradation of sev-eral insecticides, as pointed out in previous publica-tions [9–13], but also increased the crop yields. This, inturn, may reimburse the expenses for remediation ofpesticide-contaminated agricultural lands.

REFERENCES

1. Golovleva, L.A. and Golovlev, E.L., Uspekhi mikrobi-ologii (Advances in Microbiology), Moscow: Nauka,1980, pp. 137–179.

2. Baric, S., Insecticide Microbiology, Berlin, Heidelberg,New York: Springer, 1984, pp. 87–130.

3. Kruglov, Yu.V., Mikroflora pochvy i pestitsidy (SoilMicroflora and Pesticides), Moscow: Agropromizdat,1991.

4. Barles, R.W., Daughton, C.G., and Hsieh, D.P.H., Arch.Environ. Contamin. Toxicol., 1979, vol. 8, no. 6,pp. 647–660.

5. Skladny, G.J. and Metting, F.B., Soil Microbial Ecology,New York: Marcel Deccer, 1993, pp. 483–513.

6. Vasil’eva, G.K., Surovtseva, E.G., and Belousov, V.V.,Mikrobiologiya, 1994, vol. 63, no. 1, pp. 129–144.

7. Kuritz, T. and Wolk, C.D., Appl. Environ. Microbiol.,1995, vol. 61, no. 1, pp. 234–238.

8. Pfender, W.F., J. Environ. Quality, 1996, vol. 25, no. 6,pp. 1256–1260.

9. Nuikina, I.R., Krugolov, Yu.V., and Balashova, E.K.,USSR Inventor’s Certificate no. 1250572, Byull. Izo-bret., 1986, no. 30, p. 74.

Table 6. The growth of seedlings and dynamics of tomato crop yields (cultivar Grain) during plant cultivation on pressurizedpeach briquettes inoculated by microorganisms (large-scale experimental set)

Experimental setTomato seedlings Tomato crop yields, kg per 100 plants

height, mm mass of one plant, g 7th day 14th day 21st day

Control set 200 280 15 30 130

Bac. megaterium 460 510 23 50 150

E. nigrum 300 420 19 49 142

P0.95 % 7.8 7.2

Table 7. Effects of microorganisms on the development of cucumber and pepper test-plants (substrate 2)

Plant, parameter Control set Bac. megaterium E. nigrum P0.95 %

Cucumber

plant height, mm 95 196 244 8.0

mass of ten plants, g 63 127 172 8.4

mass of roots, g 0.7 4.8 10.0 9.1

Pepper

plant height, mm 150 200 246 3.5

mass of ten plants, g 69 101 197 9.3

326

APPLIED BIOCHEMISTRY AND MICROBIOLOGY Vol. 37 No. 3 2001

LISINA et al.

10. Lisina, T.O., Gavrilova E.A., Mikrobiologicheskiemetody zashchity okruzhayushchei sredy (Microbial Pro-tection of Environment), Pushchino: Izd. NTsBI ANSSSR, 1988, p. 37.

11. Garan’kina, N.G. and Pavlenko, V.V., Mikrobiolog-icheskie metody zashchity okruzhayushchei sredy(Microbial Protection of Environment), Pushchino: Izd.NTsBI AN SSSR, 1988, p. 23.

12. Lisina, T.O., Gavrilova, E.A., and Kruglov, Yu.V., TrudyVNITs Sel’skokhoz. Mikrobiol., 1990, vol. 60, pp. 100–105.

13. Gavrilova, E.A., Lisina, T.O., and Kruglov, Yu.V., USSRInventor’s Certificate no. 1735359, Byull. Izobret., 1992,no. 19, p. 69.

14. Dospekhov, B.A., Metodika polevogo opyta (s osnovamistatisticheskoi obrabotki) (Field Experiment Principlesand Statistics), Moscow: Agropromizdat, 1985.

15. Kaspirova, T.A., Dokl. Vses. Akad. Sel’skokhoz. Nauk,1981, no. 12, pp. 17–18.

16. Grodzinskii, A.M., Allelopatiya rastenii i pochvoutomle-nie (Plant Allelopathy and Soils Fatigue), Kiev: NaukovaDumka, 1991.

17. Tsareva, R.I., Khimizm torfyanoi pochvy i rost rastenii(Chemistry of Peat Soils and Plant Growth), Minsk:Nauka i Tekhnika, 1976.

18. Orlov, D.S., Gumusovye kisloty pochv i obshchaya teor-iya gumusoobrazovaniya (Soil Humus Acids and Theoryof Humus Formation), Moscow: Mosk. Gos. Univ.,1990.

19. Marshunova, G.N., Rol’ mikroorganizmov v plodorodiipochv i povyshenii effektivnosti udobrenii (Role ofMicroorganisms in Soil Fecundity and Increase in Effec-tiveness of Fertilizers), Leningrad: Kolos, 1964, pp. 67–74.

20. Berestetskii, O.A., Fitotoksicheskie svoistva pochven-nykh mikroorganizmov (Phytotoxic Properties of SoilMicroorganisms), Leningrad: Izd-vo VASKhNIL, 1978,pp. 7–30.