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ELS EVI E R Resources, Conservation and Recycling 18 (1996) 79-85 ....

Novel inoculants for an environmentally-friendly crop production

N. Kozyrovska a'*, G. Kovtunovych a, E. Gromosova b, P. Kuharchuk c, V. Kordyum a

alnstitute for Molecular Biology and Genetics of the National Academy of Sciences, Zabolotnogo, 150, Kyiv 252143, Ukraine

bInstitute for Microbology and Virology of the National Academy of Sciences, Zabolotnogo, 154, Kyiv 252143, Ukraine

Clnstitute for Agriculture of the Ukrainian Agricultural Academy of Sciences, Chabany 252205, Kyiv Region, Ukraine

Abstract

The high input of agrochemicals into soils is a major agricultural technique for enhancement of plant crop production, but it is also an environmental hazard. Concern over health hazards related to agrochemicals, especially on radioactively polluted territories, as well as economic problems, have promoted fundamental research to search for new agrobiotech- nologies. Nonsymbiotic plant growth-promoting Rhizobacteria (PGPR) are often used as inoculants; however, they are not as effective as endophytic bacteria. These bacteria have the advantage of living within the plant tissue, protecting the plant from superinfection by soil bacteria, and recolonizing the plant surface after some stress situations in the soil. Although the use of beneficial microorganisms is not a new idea, it is an idea that is not heavily utilized. Application of inoculants designed on the base of competitive endophytic bacteria may be a full or partial alternative to agrochemicals, and can diminish the level of penetration and accumulation of heavy metals and radionuclides inside plants. This study evaluates the input of the association of the bacteria, isolated from the plant interior, in the crop of corn (Zea mays) and develops inoculants on their base. Nitrogen-fixing Klebsiella oxytoca VN13 along with Xanthomonas maltophilia VN12 were found to be useful bacteria, the former capable of excreting auxins and antimicrobial substances, the latter promoting assimilation of soil phosphorous by the plants. As the association of the two bacteria has

* Corresponding author. Tel: + 38 044 2660789; fax: + 38 044 2660759; e-mail: [email protected].

0921-3449/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved PII S092 1-3449(96)01 1 70-6

80 N. Kozyrovska et al. / Resources, Conservation and Recycling 18 (1996) 79-85

been chosen as an effective endophytic system for the inoculant development, the two are mixed to form an inoculant 'Duet' which is directly inoculated into seeds. Corn inoculated with this 'Duet' produced greater yields, and possessed a greater percentage of protein. In addition, experiments performed in Chernobyl, showed that the 'Duet' could protect the plant from radionuclides penetration. It is suggested that a novel inoculant, 'Kleps', be applied, as the formulation is simple, it is inexpensive, it can be produced on a large scale, and it can be stored for a long period of time in a relatively small volume. It is clear that simple formulations of novel inoculants can be designed on the base of the competitive endophytic bacteria for an environmentally friendly crop production on poor and polluted territories as an alternative to the use of agrochemicals. Copyright © 1996 Elsevier Science B.V.

Keywords: Endophyte; Inoculant; Crop production

I. Introduction

The high input of agrochemicals into soils is a major agricultural technique for enhancement of plant crop production. Its hazard to the environment is well known: overfertilization leads to pollution and eutrophication. Another factors are energy costs for production and distribution. Plant rhizosphere bacteria are now considered as a valuable means to reduce the input of chemicals. In Ukraine, chemical inputs in agriculture are considerably lower than in the Western world for economical reasons. However, both increasing concern on health hazard of agrochemicals, especially on the radioactively polluted territories, and economical problems have promoted fundamental research in the area of alternative agriculture and search of new agrobiotecnologies.

Nonsymbiotic plant growth-promoting Rhizobacteria (PGPR) are long time in use as inoculants, however, colonization of the plant roots with PGPR is often problematic, because application of inoculant should be at the precise time needed for the plant. Moreover, being in natural biocenosis, they lose competitiveness over endemic bacteria. The endophytic bacteria may derive significant competitive advantage over PGPR from their close contact with plants [1]. Living within the plant tissue, they may protect the plant from superinfection by soil bacteria. Furthermore, localization within the plant may provide endophytic bacteria with the ability to recolonize the plant surface alter some stress situations in the soil.

The idea to use beneficial microorganisms as inoculants appeared long ago; however, the idea to use the endophytic bacteria for the non-leguminous plants inoculation guides the work of a few groups from Brazil, Germany, China, and Ukraine for the last ten years [2 9]. Application of inoculants designed on the base of competitive endophytic bacteria, possessing a set of beneficial for plants charac- ters, might be, on one hand, a full or partial alternative to agrochemicals. On the other hand, bacteria, highly colonizing plants, can diminish the level of penetration and accumulation of heavy metals and radionuclides inside plants. The objective of

N. Kozyrovska et al. / Resources, Conservation and Recycling 18 (1996) 79-85 81

this study was to evaluate the input of the association of bacteria, isolated from the plant interior, in the crop of corn (Zea mays) and to develop inoculants on their base.

2. Results and discussion

Nitrogen-fixing Klebsiella oxytoca VN13 along with Xanthomonas maltophilia VN12 were both isolated from the rice root interior in Vietnam. The electron micrographs of rice, rye, corn, and tobacco inoculated with bacteria showed their ability to penetrate to the plant interior. Bacteria were localized in cells of the peripheral layer of the root tissue, in intercellular space, and xylem [8,9]. Studies on physiological properties of these bacteria from the association have shown that one of them, the nitrogen-fixing strain, K. oxytoca VN13, is also capable to excrete auxins [10] and antimicrobial substances. Another partner of association, X. maltophilia VNI2, promotes assimilation of soil phosphorus by the plants. Monitor- ing the survival of bacteria in the plant rhizosphere with help of bioluminescence- based technique showed their presence on the roots during the plant vegetative period from May to September [11]. The association of bacteria described has been chosen as an effective endophytic system for the inoculant development.

2.1. The inoculant 'Duet'

The simplest method of the association of K. oxytoca VN13 and X. malthophilia VN12 ('Duet') application was direct inoculation of seeds with a mixture of bacterial cultures. A set of microplot experiments have been conducted to test input of the bacterial association in crop of the corn grown without nitrogen fertilizers implementation as well as on different dosages of nitrogen (30, 90, and 120 kg per hectare). Experiments have been performed in the Kyiv region (Kopylove) in the period 1992-1995. The plots (40 m 2) used in four replications have been randomly situated. The characteristics of the soil in Kopylove: organic matter, 1.03%; PzO s, 7.95; K20, 10.08 mg in 100 g soil; pH, 4.3. The data on corn yield given are means of four replications. Analysis of variance was performed to test the signification of some observed differences in weight of grain (14% of humidity), protein, nitrates, and phosphorus content in the air-dried grain (P = 0.05).

Results represented on Fig. 1 show the increased yield of corn inoculated with the 'Duet' and grown on different background of nitrogen as compared to control (without inoculation). Inoculation of crop with associated bacteria is nearly equal the use of 90 kg N/ha. In accompany with sustainable agricultural techniques the effectiveness of the biopreparation is more than the input of 120 kg N/ha. Inoculation of corn seeds by bacterial mixture ('Duet') increased the protein content in grain by 10 14% (Fig. 2). The protein content in the corn grain was maximal when a start dosage of fertilizer had been used (30 kg N/ha). In this case, content of nitrates did diminish by 7% as compared to non-inoculated variant of corn grown on the same background of a nitrogen fertilizer (data not shown).


- . % - . - . - ,

t/ha

KLEPS

DUET

No N 3 0 ~ ~ 0 9 0 control

N120

Fig. 1. Average yield of corn is dependent on seed inoculation and fertilizer input (1992-1995, Kyiv region).

Experiments performed in Chernobyl on the radioactively polluted soil showed that the 'Duet ' could protect the plant from radionuclides penetration; at least bacteria decreased two-fold the level of Cs-137 accumulation in the corn biomass (Table 1).

9- ~ :2-'-'-'"

...... grain ~. :!i:!::i::: 'fl+ +

N90

Fig. 2. Average protein content in air-dried corn grain (1992-1994, Kyiv region).

N. Kozyrovska et al. / Resources, Conservation and Recycling 18 (1996) 79-85

Table 1 Accumulation of cesium-137 in corn inoculated with 'Duet'

83

Inoculant/ Coefficient of Cs-137 transition into corn Amount of Cs-137 in the corn crop biomass , Bq/kg, (Bq/kg)

Duet Roots 0.11 910 Shoots 0.03 285

Control Roots 0.29 2350 Shoots 0.06 445

2.2. The inoculant 'Kleps'

Direct seeds inoculation with bacterial culture is a world-wide common practice, easy to use and inexpensive; however, a liquid inoculant cannot be stored for long time. Various solid-phase bacterial inoculants (mainly peat variants and polymer gels for entrapping bacteria or fungi) are in use during the last decade [12-15]. Mineral microgranules, containing bacterial mass, can be used as biofertilizer, too [16-18]. Economical reasons stimulate a search of an inexpensive matrix for the inoculant. A novel carrier for the 'Duet ' bacterial association has been designed on the base of microbial exopolysaccharide, mucilan, produced by Bacillus mucilaginosus [19]. Mucilan has been used as both a carrier and a protective matrix for bacteria.

Novel inoculant, 'Kleps', combines useful properties of both bacteria of the association, providing the plant with biological nitrogen, phytohormones, fungi- cides, and a stimulating and protective activity of the carrier, mucilan. The inoculant 'Kleps' can be used for pre-treatment of the spring and winter cereals seeds as well as horticultural ones. Three-year field trials in three geographical regions of the Ukraine showed spectacular increase of the corn yield (see Fig. 1). The study of survival of bacteria marked with the lux genes as described earlier [11] showed that the population of the Lux + bacteria did not decrease after 3-month storage of 'Kleps' at a room temperature. The formulation of 'Kleps' is simple, and due to this it is inexpensive. It may be produced on a large scale by the industry and can be stored for long periods in a relatively small volume.

3. Conclusions

The endophytic bacteria are considered as a valuable means to reduce the input of agrochemicals. The advantages of these bacteria are connected with plant tissue localization: (i) competitiveness; (ii) recolonization ability; (iii) delivery of biologi- cally active substances to the plant. Simple formulations of novel inoculants have been designed on the base of the competitive endophytic bacteria for an environ-


menta l l y - f r i end ly c rop p r o d u c t i o n o n p o o r a n d p o l l u t e d ter r i tor ies as a l t e rna t ives to

the use o f agrochemica ls .

Acknowledgements

W e express a p p r e c i a t i o n to Bor is So roch insk i for expe r imen t s p e r f o r m e d in C h e r n o b y l , to L id i a H a l d a - A l i j a ( U S A ) for a cri t ical r e ad ing o f the m a n u s c r i p t , a n d to T a m a r a V o z n y u k a n d T a n y a S t re l t ch ina for t echnica l assis tance. Th i s s tudy was s u p p o r t e d by U k r a i n i a n State C o m m i t t e e o n Science a n d T e c h n o l o g i e s a n d R & D Cen t r e ' E n e r g i y a ' (Kyiv) .

References

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