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UsingPlantGrowthPromotingRhizobacteriatoImprovePotatoProductionandPlantHealth

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USING PLANT GROWTH PROMOTING RHIZOBACTERIA TO IMPROVE POTATO PRODUCTION AND PLANT HEALTH

A. OSWALD1, P. CALVO, J. SANCHEZ1, D. ZUÑIGA2 1 International Potato Center (CIP), Av. La Molina 1895, Lima 12, Perú, a.oswald@cgiar.org, tel.: +51-1-3496017, fax: +51-1-31753262 Universidad Nacional Agraria La Molina, Av. La Molina, Lima 12, Peru, dzuniga@lamolina.edu.pe, Tel: +51-1-7995788, fax: +51-1- 3496015

Abstract

Plant Growth Promoting Rhizobacteria (PGPR) have shown their potential in various crops to improve yield and plant health. In this respect PGPR could be developed into an alternative inexpensive bio-fertilizer to increase potato yields of small scale subsistence farmers and/or to improve organic crop production systems in the Central Andean Highlands of Peru. In 2005/06 commercially available PGPR were applied in field trails with potatoes, resulting in yield increases of 20-30% and partial control of Rhizoctonia solani. Increased yields were due to greater sink size and sink strength of the inoculated potato crop. Populations of Azotobacter and Bacillus subtilis in the rhizosphere of the potato were greater during the entire growth period as compared with non-inoculated control potato plants. Based on these results PGPR research will be continued and native bacteria in the major potato- growing regions of Peru will be sampled, identified, tested and selected. PGPR shows a clear potential to increase nutrient use efficiency of potato that could be developed as an important element in low and high input crop production systems.

Introduction

Farmers in the Central Andean Highlands of Peru cultivate potatoes as their staple food crop. Potato may also be their most important cash crop. Farmers in the area employ a wide variety of cropping systems, comprising high input systems with fertilizer applications of up to 500 kg NPK ha-1 and frequent pesticide use or subsistence systems with low organic manure applications and almost no pesticides. Yield levels are medium to low, ranging between 15 and 20 t ha-1 for high input and 5 to 8 t ha-1 for low input systems indicating low soil fertility and low nutrient use efficiency (Davies Jr. et al., 2005). In these variable agro- ecological conditions and diverse cropping systems, beneficial soil microorganisms could play an important role in improving crop yields and reducing disease incidence. Root- colonizing bacteria that exert beneficial effects on plant development via direct or indirect mechanisms have been defined as plant growth promoting rhizobacteria (PGPR) (Nelson, 2004). Among others these mechanisms consist of fixation of atmospheric N, solubilization of P and Fe, synthesis of plant hormones and antibiotics to promote plant growth and control deleterious fungi (Glick, 1995). Hence, the use of PGPR could improve nutrient use efficiency by the potato crop, reducing fertilizer applications, and partially control soil borne diseases, which are affecting tuber yield and tuber quality. These bio-fertilizers would help to sustain environmental health and soil productivity and reduce costs of crop production (Orhan et al., 2006). The prospect of using beneficial microorganisms to increase plant

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growth has shown considerable promise in laboratory and greenhouse studies, but responses have been variable in the field (Bowen and Rovira, 1999). This variability has often been attributed to the inability of PGPR to colonize plant roots, compete with indigenous microorganisms and adapt to the specific environmental conditions (soil pH, temperature, nutrient supply, etc.) (Benizri et al., 2001). For those reasons this study used a field and laboratory approach simultaneously. In the first year commercially available PGPR were tested in field trials with a potato crop in contrasting environments to evaluate their ability to promote plant growth and increase tuber yield. A second objective of these trials was to study the response of different genetic material, such as Solanum tuberosum x S. andigena or S. andigena commercial varieties and indigenous landraces, to inoculation with rhizobacteria. The results will be reported in this paper. At the same time native bacteria were sampled from the rhizosphere of potato plants in different regions of the highlands of Peru and subjected to various in vitro tests and pot trials with the aim of pre-selecting suitable organisms for further field studies, assuming that these PGPR will be better adapted to the environmental conditions of the highlands.

Materials and methods

In the rainy season (November–April) 2005/06 two field experiments were conducted with PGPR and mycorrhiza in potato crop at two sites in the Central Andean Highlands of Peru (Table 1).

In Aymara a field experiment was implemented in a farmers’ field after a fallow period of 5 years. The trial layout was a completely randomized block design (CRBD) with 2 factors and 4 replications. The plots had 9 rows with 15 potato plants each; row spacing was 0.9 m and intra-row spacing 0.3 m. The harvest area was 17.55 m2, comprising 7 central rows and 13 plants per row, leaving the first and last plant as borders. The treatments consisted of 3 Peruvian potato varieties – Yungay, Putis, Queccorani – adapted to the agro-ecological conditions of this region as factor A and 5 fertilizer treatments as factor B. The treatments were as follows: 5 t manure ha-1, Azotobacter sp. + 5 t manure ha-1, Bacillus subtilis + 5 t manure ha-1, Glomus fasiculatum (mycorrhiza) + 5 t manure ha-1, 120 kg N, 100 kg P, 100 kg K ha-1 (ammonium nitrate, triple superphosphate, potassium chloride) + 5 t manure ha-1.

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The microbial products were applied at higher than recommended rates to increase success of inoculation under these difficult environmental conditions (Table 2). At planting, hilling-up and flowering the entire bacterial populations of Azotobacter spp. and Bacillus spp. were determined (not separating the specific applied bacteria from the native soil bacteria of the same genus), taking soil and rhizosphere samples from potato plants and processing and analyzing these samples in the laboratory using glucose-tryptone-agar for isolating Bacillus spp. and nitrogen-free culture broth for Azotobacter spp. (Bergey, 1986, Merck, 1994). Mycorrhiza concentrations and infestation were not assessed as the team did not have the knowledge for this type of investigation.

Table 2. Bio-products and application rates of three microorganisms in potato

The second trial was installed on a research farm in Huancayo. The design was a CRBD with 2 factors and 3 replications. A total of 23 potato varieties, mainly Peruvian potato landraces, were planted and fertilized either with 5 t manure ha-1, as the control, with Azotobacter + 5 t manure ha-1, or with B. subtilis + 5 t manure ha-1. The treatments were planted in micro- plots consisting of 3 plants. All plants were harvested for tuber yield. The application rates for PGPR were the same as in the first trial. Mycorrhiza was not used because no inoculum was available. Both trials were protected against pests and diseases as necessary. The insecticide Fipronil (Regent) was used against Epitrix yanazara and Premnotrypes sp. and the fungicides Mancoceb (80%) (Dithane) and Propineb & Cymoxanil (Fitoraz) for preventive and curative applications against Phythophtera infestans. Most potato varieties were harvested after 120 to 130 days. At harvest the potato tubers were graded as commercial (> 80 g), non-commercial (< 80 g) and damaged tubers and their weight and number recorded. The damaged tubers were further separated in groups with and without Rhizoctonia solani infestation. Yield data were subjected to analysis of variance (ANOVA). Means were separated using Duncan’s Multiple Range Test. Percentage data were transformed using square root transformation and then subjected to ANOVA (Gomez and Gomez, 1984).

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Results and discussion

For the field trial in Aymara the bacterial populations of Bacillus spp. and Azotobacter spp. were monitored from one month after planting until flowering, which was about four months after planting (Fig. 1a & 1b). For Bacillus spp. the populations decreased slightly from the first to the second sampling and then remained constant until flowering. For Azotobacter spp. the same pattern could be observed except for the populations with the variety Queccorani, which decreased more than 10 fold between the first and last sampling. Generally, bacterial populations were higher in treatments with PGPR applied over the entire growth period. They were similar in the rhizosphere of potatoes of the varieties Yungay and Putis but clearly higher for the variety Queccorani. Specific responses of plant genotype–bacteria have been recorded for tomato, wheat and other crops (Romero et al., 2003). This might be due to the better growth conditions that the bacteria encountered in the rhizosphere of this variety and needs further investigation. However, the better colonization of the roots of the variety Queccorani had no effect on tuber yield or plant growth as no difference in yield between varieties could be observed (Table 3).

Figure 1a and 1b. Population dynamics of Bacillus spp. and Azotobacter spp. in three different potato varieties, Aymara, Huancavelica, rainy season 2005/06

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Tuber yields were low but had an acceptable level considering the low organic manure application and the agro-ecological conditions with low temperatures, frequent drought spells of 1-2 weeks and high soil acidity. All varieties responded similarly to the different treatments; no interactions were observed. The highest yields were achieved with a mixture of organic and inorganic fertilization, more than doubling the yields of the control with application of 5 t of manure ha-1. Microorganisms increased tuber yields by up to 25% compared to the control with manure only, the most effective being Azotobacter and mycorrhiza. The yield increase was due to more tubers being produced per plant and a higher rate of commercial tubers (data not shown). The varieties Putis and Queccorani had 15–35% more tubers, if microorganisms were applied, as compared to the control treatment, while NPK fertilization improved tuber numbers by up to 50%. The variety Yungay had relatively staple tuber numbers and no effect of microorganisms could be observed. Also the number of commercial tubers (tuber > 80g of weight) increased with application of Azotobacter, mycorrhiza and inorganic fertilizer but not with B. subtilis. Therefore, the proportion of commercial tuber weight of the entire tuber weight increases from 40 % for the control and the treatment with B. subtilis to 43 and 47% for mycorriza and Azotobacter respectively, and up to 54% for the NPK treatment.

Table 3. Tuber yields in t ha-1 of three potato varieties fertilized organically and inorganically and treated with three different microorganisms, Aymara, Huancavelica, rainy season 2005/06

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Tuber yields of the trial with 23 potato varieties ranged between 0.6 kg m-² and 6.6 kg m-². Grouping the varieties according to their response to the treatments with PGPR showed three principal patterns: - Tuber yields were increased by application of Azotobacter and B. subtilis – 8 varieties; - Only one of the two PGPR increased yields (mainly Azotobacter) – 5 varieties; - The application of B. subtilis and Azotobacter did not increase yields – 10 varieties.

In Fig. 2 the tuber yields of the same varieties of the first and second group are shown to illustrate the beneficial effect of PGPR on potato growth. Again these results show that the effect of PGPR on tuber yield is influenced by the potato variety and interactions between the two organisms.Yield increases were achieved by a greater number of tubers produced per plant and especially in the case of inoculation with Azotobacter, as well as by an increase of average tuber size (data not shown). At harvest the infestation of tubers with Rhizoctonia solani was recorded, as some PGPR give also partial protection against this fungus (product description FZB24). Infestation ranged between 3 and 12% and no significant differences could be observed among varieties, indicating a relatively even spread of the disease. However, on average (of all varieties) 7.6% of the tubers of the control treatments were infected; equivalent numbers were only 2.3% with inoculation of B. subtilis and 3.9% with inoculation of Azotobacter. Although these microorganisms were collected and selected for use in very different climatic and agro-ecological conditions (Germany, Peru, Bolivian lowlands), they proved their effectiveness in stimulating plant growth and partially inhibiting soil-borne diseases in the Central Andean Highlands as well. Under the specific conditions of

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the field trials (Table 1) great differences in soil pH, soil organic matter content or aluminum concentrations between the two locations did not affect their plant growth-promoting ability. This shows either a strong adaptability of the bacteria to diverse agro-ecological conditions or rather that plants are able to provide similar growth conditions within their rhizosphere to support bacterial (and fungal) development.

Figure 2. Tuber yields of eight potato varieties inoculated with Azotobacter and Bacillus subtilis, Huancavelica, rainy season 2005/06

Based on these results and as a second step in the evaluation of PGPR, native rhizobacteria of potato were sampled in different locations with distinct agro-ecological conditions of the Central Andean Highlands to be isolated identified and tested with standard in vitro procedures. The best ones are currently being investigated in pot and field trials with the aim of finding more effective bacteria than the commercial ones that can be successfully developed into a ‘bio-fertilizer’. A ‘bio-fertilizer’ in this respect would not be defined as a nutrient-carrying agent but rather as a ‘catalytic promoter’ that improves nutrient use efficiency and general growth conditions of plants, not substituting any external nutrient supply but increasing the uptake of applied fertilizers or natural soil nutrients. Additionally, PGPR also have the potential to increase plant health by suppressing deleterious microorganisms, thus making them a valuable factor in an integral concept of soil fertility and soil health management.

Acknowledgement

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The authors thank the support of the International Potato Center and the Universidad Nacional Agraria La Molina, Lima, Peru, to implement this study and acknowledge the financial assistance of the Papa Andina Initiative.

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References

Benizri, E., Baudoin, E. & Guckert, A. 2001. Root colonization by inoculated plant growth promoting rhizobacteria. Biocontrol Sci. Technol., 11, 557-574.

Bergey 1986. Bergey’s manual of systematic bacteriology. Gram positive bacteria other than Actinomycetes. Holt, J.G. (ed.). Springer, New-York, pp. 630.

Bowen, G.D. & Rovira, A.D. 1999. The rhizosphere and its management to improve plant growth. Adv. Agron., 66, 1-102. Davies Jr., F.T., Calderon, C.M., Huaman, Z. & Gomez, R. 2005. Influence of a flavonoid on mycorrhizal acitivity in the highlands of Peru. Sci. Hort. 106, 318-329.

Glick, B. R. 1995. The enhancement of plant growth by free-living bacteria. Can. J. Microbiol. 41, 109-117.

Gomez, K.A. & Gomez, A.A. 1984. Statistical Procedures for Agricultural Research. An International Rice Research Book. Wiley-Interscience publication, John Wiley & Sons, New York, pp. 680.

Merck 1994. Manuam de medios de cultivo. E. Merck, Darmstadt, Germany, 364 p.

Nelson, L.M. 2004. Plant growth promoting rhizobacteria: prospects for new inoculants. Plant Manag. Netw., www.plantmanagementnetwork.org/pub/cm/review/2004/rhizobacteria/

Orhan, E., Esitken, A., Ercisli, S., Turan, M. & Sahin, F. 2006. Effects of plant growth promoting rhizobacteria on yield, growth and nutrient contents in organically grown raspberry. Sci. Hort. 111, 38-43.

Romero, A.M., Correa, O.S., Moccia, S. & Rivas, J.G. 2003. Effect of Azospirrilum-mediated plant growth promotion on the development of bacterial diseases on fresh-market and cherry tomato. J. Appl.Microbiol., 95, 832-838.