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Microbiological Improvement of Root Health, Growth, and Yield of Strawberry


Final Report - September 2002, (Updated May 2003)


Principal Investigator:
John M. Duniway, Professor
Department of Plant Pathology
University of California, Davis
One Shields Avenue
Davis, CA 95616-8680
Telephone: 530-752-0324
Email: jmduniway@ucdavis.edu
Fax: 530-752-5674

Cooperators:
Luis Guerrero and Christopher Winterbottom
California Strawberry Commission
P.O. Box 269
Watsonville, CA 95077-0269
Phone: 831-724-1301
Email: LGuerrero@calstrawberry.org
Fax: 831-724-0660

Kirk Larson
Department of Pomology
University of California, Davis
UC South Coast
Res. & Ed. Ctr.
7601 Irvine Blvd.
Irvine, CA 92718
Phone: 949-857-0136
Email: kdlarson@ucdavis.edu
Fax: 949-653-1800

Project Locations
UC Davis Campus, Yolo Co., Monterey Bay Academy, Watsonville, Santa Cruz Co., and UC South Coast Research and Education Center, Irvine, Orange Co.

Commodity:
Strawberry

Funding:

  SAREP Direct Cost Matching from the California SAREP Direct Cost Strawberry Commission
1999-2000 $36,670 $4,680 CSC Project to PI
12,000 CSC Field Expenses
2000-2001 $40,260 $4,680 CSC Project to PI
12,000 CSC Field Expenses
2001-2002 $41,850 $4,680 CSC Project to PI
12,000 CSC Field Expenses

 

Table of Contents

Objectives
Summary
Specific Results
Potential Benefits/Impacts on Agriculture
Dissemination of Findings
Figures

Objectives

The research objective was to find and effectively deploy microorganisms to improve root health, growth, and yield of strawberry plants without soil fumigation or with less than optimum soil fumigation treatments. While no individual microorganism or combination of beneficial microorganisms is likely to reproduce the large yield increases obtained by methyl bromide/ chloropicrin fumigation of soil, evidence was found that inoculations with specific microorganisms are likely to increase yield significantly. These increases are most likely to be useful when combined with alternatives to methyl bromide, including fumigants other than methyl bromide. Candidate microorganisms are available commercially, but more likely to succeed are microorganisms isolated recently from roots of strawberry growing in fumigated soils in California. The approach was to use these microorganisms, which were found to promote growth of strawberry plants in the greenhouse, to inoculate plants grown for berry production in the field. Methods of field application were researched and resulting growth and yield responses of strawberry measured relative to those obtained by normal farming practices with and without fumigation. The educational objectives were to help demonstrate mechanisms by which strawberry responds to soil fumigation and to scientifically explore, with grower involvement, the feasibility of using biological agents to help improve strawberry health and yield.

Summary

We continued to sample field sites throughout the project period for additional isolates of rhizosphere bacteria and to test their effects on the growth and health of strawberry in the greenhouse and growth chambers. In the last 2 years, we improved our screening efficiency by testing bacteria for inhibition of several pathogenic fungi in culture. Several new isolates with beneficial activity were found and some were tested in the field.

In each of the 3 years of the grant, several bacteria (and sometimes specific fungi) were used to inoculate strawberry plants in replicated field experiments. These were done at the Monterey Bay Academy (MBA), Watsonville, and at the U.C. South Coast Research and Education Center (SCREC), Irvine. Three bed fumigation treatments were applied each year at MBA, i.e., a standard rate of methyl bromide/chloropicrin (MBC), a low rate of chloropicrin (Pic), and not fumigated. Plants were root-dip inoculated at transplanting and some were reinoculated periodically during crop growth. While MBC fumigation approximately doubled strawberry yields, none of the inoculation treatments increased yields significantly in MBC-treated and very few did so in nontreated soil. In contrast, several of the bacteria tested increased yields in soil treated with a low rate of Pic at MBA, and some of these increases were statistically significant. Reinoculation during crop growth did not enhance the effects of the bacteria. Additional experiments were done in the last 2 years at MBA using the variety Aromas and nonfumigated soils. A few of the bacteria tested reduced the incidence of Verticillium wilt in 2001 and two isolates increased yields in 2002. Aromas appears to be more responsive to bacterial inoculations than Selva.

Sections of the ground used in 1999-2000 at SCREC were broadcast fumigated with MBC or were left untreated. Bare-root Camarosa runner plants were obtained from a high elevation nursery and Camarosa plug plants were propagated by Kirk Larson. In 2000-01, ground at SCREC, which was new to strawberries, was bed fumigated with MBC, Pic, or not treated, and in 2001-02 beds were fumigated with MBC, metam sodium, or were not treated. The field used in 2001-02 had a history of strawberry production. Rhizosphere bacteria were used to inoculate bare-root transplants only at the time of planting. The effects of the soil fumigation and inoculation treatments on plant size at SCREC were variable, but fumigation generally increased yields significantly on ground with a history of strawberries. One bacterium increased growth significantly in Pic- and non-treated soils, while two did so following metam sodium treatment of soil. The use of plug plants in 1999-2000 had only small and variable benefits relative to bare-root transplants.

The main aspects of these experiments are being repeated at MBA in 2002-03 with additional bacterial isolates from strawberry and support from the California Strawberry Commission. In the 2002-03 crop cycle, we have found that marked strains (antibiotic resistant) do colonize soil and roots following inoculations, with high numbers on both older and new roots up to 2 months after inoculation. Dispersal of marked strains has appeared vertically downward from the points (tested at about 10 cm). At 5 months, plants were nearly fully grown and there were still fairly high numbers of inoculated bacteria on roots at shallow depths, but low numbers deeper in soil. There was no spread laterally at the 10 cm distance tested. These plots are still being harvested, and net yield effects are yet to be determined.

Specific Results

Microbial Isolates - Many studies have shown that root colonization by specific bacteria or fungi can give general increases in plant growth, yield, and/or measurable control of known root pathogens (2, 3, 11-14, 19). While most of the research has been directed at the effects of individual rhizobacteria on a specific pathogen, individual isolates of rhizobacteria are being found to have broader spectrums of activity against multiple plant pathogens (12, 19). In addition, rhizobacteria have recently been found to induce systemic resistance in plants, i.e., to reduce general tissue susceptibility to various pathogens throughout the plant (18). A number of bacteria and fungi have been inoculated to seeds or transplants to improved plant growth, yield, or disease control in natural soils, and some of these agents have been commercialized (2, 3, 11, 14, 19). Commercial strains of bacteria have recently been tried on strawberry in California, but were of little benefit in a field situation where soil fumigation also gave small increases in yield (1).

The PI and associates have been researching microbiological differences associated with the enhanced growth and productivity of strawberries in soils fumigated with methyl bromide/ chloropicrin where the response is not due to control of known, major pathogens (4-10, 16, 17). Briefly, strawberry plants in fumigated soils consistently had much higher root length densities and fewer dark roots than plants in nonfumigated soils. Fungi, including species of Cylindrocarpon, Pythium, and Rhizoctonia which we find to be damaging to strawberry roots, are isolated significantly less often from roots in fumigated than in nonfumigated soils. Populations of fluorescent pseudomonades in soil increase quickly following fumigation to be 10-1000 fold higher than in nonfumigated soils 10 days to 9 months after fumigation. Predominant isolates of fluorescent pseudomonades from the rhizosphere were tested for effects on strawberry growth in natural field soil in the greenhouse. The effects of individual isolates ranged from beneficial (increased shoot and root dry weights up to 72% and 162%, respectively) to deleterious (about 20% shoot or root reduction). Pseudomonas fluorescens, P. putida and P. chlororaphis were among the most predominant and beneficial rhizobacteria tested. The results suggest that reductions in deleterious fungi and increases in beneficial fluorescent pseudomonades contribute to the enhanced growth response of strawberry to soil fumigation with methyl bromide/chloropicrin.

The main approach taken in the project reported here was to use beneficial microorganisms isolated from strawberries in fumigated soils as inoculants in field experiments to determine the feasibility of using microorganisms beneficial to root health and growth in strawberry production. At the start of this project, we had a collection of candidate bacteria isolated previously from several field sites where strawberries were grown in non-fumigated and fumigated soils with large growth and/or yield responses to fumigation. We continued to collect new candidate isolates throughout the course of this 3-year project. Replicate plants were dug from the soil to a depth of 20 cm and brought to the laboratory. Young roots were carefully lifted from the soil and sub-sampled for visual assessments of root health and transfer to buffer on a shaker. Rhizosphere soil in buffer was subsequently used for dilution plating on three media, colonies enumerated, and random samples of each colony type transferred for pure culture. Bacteria and fungi are also isolated from 5 mm root segments after washing in buffer.

Screening Isolates for Activity - Many individual bacteria were screened for their effects on the growth and health of strawberry plants using a natural soil in the greenhouse or growth chamber. Individual bacteria, kept at -80 C, were grown in TSB and used to root-dip inoculate clean, 10-week-old Selva, Aromas, and/or Camarosa seedlings (5-7 replicates) as they were transplanted into natural field soil (collected where strawberry is grown without fumigation) mixed with a potting soil. Two uninoculated controls were included in each experiment, i.e., one using the natural field soil and one using heat-sterilized field soil. Four to 6 weeks after inoculation, plants were washed free of soil, root health assessed, and fresh and dry weights of roots and shoots determined. In the last 2 years of the project, individual bacteria were also screened in culture for antibiosis to Pythium, Rhizoctonia, and Cylindrocarpon isolates found to be damaging to strawberry roots in nonfumigated soil, and to the known pathogens Verticillium dahliae and Phytophthora cactorum (9, 10, 17). We found that pre-screening for antibiosis to one or more of the fungi greatly improved the frequency with which bacterial isolates were found to increase the growth of strawberry seedlings (Figure 1).

Results from a representative greenhouse experiment using recent isolates having some antibiosis are shown in Figure 2. A majority of the isolates in this experiment increased root growth measured as fresh and/or dry weight and some also increased shoot growth. Strawberry variety did not have a significant effect (data not shown), but soil sterilization sometimes increased uninoculated plant growth significantly. Promising isolates were tested further using similar methods in controlled environment chambers, and those isolates that gave repeatable growth benefits in the growth chamber were retained for further testing and identification by fatty acid extraction and chromatography using the MIDI system. Those bacteria having the most favorable activities were advanced to field plot testing in the next crop cycle.

Inoculated Field Plots at Watsonville - Plots for the main experiments at MBA were farmed in each of 3 years by normal practices for strawberry production in the area on 2-row beds. They were located on soil with a past history of strawberry production where general growth and yield responses to soil fumigation were large (5-9). Three fumigation treatments were applied using a black polyethylene mulch, i.e., a standard bed fumigation with MBC, 67/33, 325 lb/a (rates per bed treated area, which is 58% of total area), bed fumigation with Pic alone at 200 lb/a, and not fumigated for the current crop. A split-split plot design was used, with main plots for soil treatments, subplots for individual organisms, and 3 randomized complete blocks. Bare-root Selva transplants were obtained from a high-elevation nursery. On the day of transplanting, roots and crowns were washed, longer roots trimmed, and 10 plants put into 200 ml of bacterial suspension (>106/ml) for 10 min. Inoculated plants were immediately planted by normal practice and 30 ml of bacterial suspension added to the soil at each plant. Drip irrigation was then applied to wet the root zones of transplants. In 1999-2000, inoculated plots were subsequently divided into two subplots, one which was reinoculated with the same bacterium approximately bimonthly during plant growth. This was done by adding 100 ml of bacterial suspension to the soil at the crown of each plant and then irrigating until the root zone was wetted. For each experimental unit, shoot growth was measured periodically as diameter of ground area covered by leaves. Berries were picked weekly starting early April through August, sorted for market quality and culls, and weighed. The incidence of known diseases, such as Verticillium wilt and Phytophthora root or crown rot, was measured.

Total yields of market quality berries obtained during 2000 are shown in Figure 3. All inoculation treatments decreased yield on MBC-treated soil, and none increased yield on non-treated soil. On Pic-treated soil, however, all the inoculations increased yield and some did so significantly. Periodic reinoculations during crop growth in 1999-2000 did not enhance the performance of any of the bacteria used and inoculations were done only once at transplanting in subsequent experiments. Results were similar in 2000-01 except that only one of the bacteria tested and one commercial isolate of the fungus Trichoderma harzianum T-22 (8, 11) increased yield on Pic-treated soil (Figure 4). However, while the inoculation treatments in non-fumigated soil did not increase total yields significantly, three of the bacterial did reduce the final incidence of plants with symptoms of Verticillium wilt (Figure 5). The experiment was terminated in August and it is likely that treatments reducing disease at that time would have enhanced yields obtained later that season using normal practices for the variety Aromas. In 2002-02 none of the bacteria used in an experiment with MBC-, Pic-, and non-treated soils at MBA increased the yields of Selva (data not shown). However, in a separate experiment using Aromas on non-fumigated soil, two of the inoculation treatments did increase total yields (Figure 6). Unlike most of the bacteria used, these specific bacteria (K3, K4) were isolated from plants other than strawberry by C I. Kado (Plant Pathology, U.C. Davis) and have wide biological activity against plant pathogenic fungi; they were also applied with a porous bead carrier. While the results obtained at MBA were variable, it appears that most of the bacterial inoculations of Selva in MBC-treated and nontreated soil reduced yields while a few of the same inoculation treatments increased yields in Pic-treated soil. The variety Aromas, however, sometimes responded favorably to bacterial inoculations in non-fumigated soils.

Inoculated Field Plots at Irvine - In cooperation with Dr. Kirk Larson, bacterial inoculations were also done in replicated field experiments at the U.C. South Coast Research and Education Center (SCREC), Irvine. The site used in 1999-2000 had a long history of strawberry production and sections were broadcast fumigated with MBC by standard practices. Two-row beds were subsequently made on separate fumigated and nonfumigated areas. These were then divided into two experiments and further subdivided into replicate blocks. Treatments were randomly assigned to plots within individual blocks. Camarosa was used throughout. Plug plants were propagated by Kirk Larson and conditioned outside at a high elevation nursery. Bare root plants were produced commercially at the same high elevation nursery. Bare root and plug plants were both inoculated and transplanted in October, 1999. Plug plants were placed in inoculum such that the roots and medium were saturated with bacterial suspension at planting. All plants were irrigated by sprinklers immediately after transplanting and periodically thereafter until sprinklers were replaced by drip irrigation. Inoculated plants were reinoculated with the same bacteria and plant sizes measured two times as the plants grew. Unfortunately, some plants were damaged by animals and Santa Ana winds shortly after transplanting, and plug plants survived somewhat better than bare root transplants. While soil fumigation approximately doubled berry yields in 2000, none of the inoculation or planting material variables modified yield significantly (Figure 7).

The main aspects of the experiments above were repeated in 2000-2001. Ground at SCREC, which was new to strawberries, was bed fumigated with MBC, Pic, or not treated. Four rhizosphere bacteria isolated from strawberry were used to inoculate bare-root transplants only at the time of planting. Bed fumigation with MBC was not very effective, but bed fumigation with Pic increased yield significantly. This may be due to the more effective distribution of Pic obtained with the drip emulsion application method, whereas the MBC was shank applied. The relatively small effects of fumigation in this experiment may also be due to the absence of prior strawberry culture for many years on this site. Inoculations with 2 bacteria, however, increased growth on nontreated soil, and one bacterium increased growth significantly on both nontreated and MBC-treated soil (data not shown). Inoculation treatment effects on yield within any one soil treatment, however, were finally small (data not shown).

A third experiment was done at SCREC in 2001-02. Three soil treatments (MBC, metam sodium @ 30g a.i./a, and not treated) were applied to ground with a history of strawberries. Yield responses to fumigation were modest and none of the bacteria increased yields on MBC- or non-treated soils (Figure 8). In contrast, several of the bacteria increased yields on soil treated with metam sodium (Figure 8).

Continuing research
Even though this SAREP project formally ended June 30, 2002, aspects of the research reported here are continuing. We are continuing to characterize newer isolates obtained in the last year and the most active of these will be taken to field experiments at MBA in 2002-03 along with some of the isolates used previously. The continuing research is supported by the California Strawberry Commission.

Potential Benefits/Impacts on Agriculture

Strawberry production in California relies heavily on pre-plant fumigation of soil with methyl bromide, usually in mixtures with chloropicrin. Currently, the most economically feasible alternatives to methyl bromide for strawberry production are other chemical fumigants, e.g., Telone with chloropicrin, chloropicrin alone, and metam sodium (Vapam). Many of the known chemical alternatives are somewhat less effective or more variable than methyl bromide (e.g. 5-9), and all have risks and some do not have full registration or public acceptance. While more research is needed to further optimize chemical alternatives to methyl bromide, the time has come to explore nonchemical options more scientifically and fully. Nonchemical approaches are needed to supplement chemical alternatives or possibly replace them in the longer term when used in integrated systems. The introduction of beneficial microorganisms with transplants is one such approach that can be combined with current practices and, in the future, with other approaches into less pesticide-intense systems. There is good scientific precedent for the approach proposed, which is reviewed elsewhere (e.g. 2, 3, 14, 19). The results obtained so far show some of the potential benefits and pitfalls of using biological agents to help replace methyl bromide. For example, the beneficial effects of inoculating strawberry with specific bacteria or fungi were variable between years and locations. Nevertheless, some of our bacteria and one commercial fungus were beneficial more often than not. Furthermore, the results obtained to date suggest that bacterial inoculations can be more effective on soils fumigated with metam sodium or low rates of chloropicrin. Some strawberry varieties (e.g. Aromas) may also be more responsive to beneficial bacteria than others (e.g. Selva). Clearly, the results of this project are still preliminary and more research is needed to know if and when beneficial bacteria might be used to advantage in commercial strawberry production.

Dissemination of Findings

Activities and findings of the grant have been or will be reported, in part or fully, at the following functions: 1999-2000

2000-01

2001-02

Literature Cited:
1. Bull, C. and Ajwa, H. 1998. Yield of strawberries inoculated with biological control agents and planted in fumigated or non-fumigated soil. Abstract 94. Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, Orlando, Florida. December 7-9.

2. Cook, R. J. 1993. Making greater use of introduced microorganisms for biological control of plant pathogens. Annu. Rev. Phytopathology 31:53-80.

3. Cook, R. J., and Baker, K. R. 1983. The Nature and Practice of Biological Control of Plant Pathogens. American Phytopathological Society, St. Paul, MN. 539p.

4. Duniway, J. M., 2002. Non-chemical alternatives used in the USA on horticultural crops. Proceedings of International Conference on Alternatives to Methyl Bromide. Sevilla, Spain, 5-8 March. Pages 258-60. http://europa.eu.int/comm/environment/ozone/conference/

5. Duniway, J. M., Gubler, W. D., and Xiao, C. L. 1997. Response of strawberry to some chemical and cultural alternatives to methyl bromide fumigation of soil under California production conditions. Abstract 21. Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, San Diego, CA. November 3-5.

6. Duniway, J. M., Hao, J. J., Dopkins, D. M., Ajwa, H., and Browne, G. T. 2000. Some chemical, cultural, and biological alternatives to methyl bromide fumigation of soil for strawberry. Abstract 9. Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, Orlando, FL. November 6-9.

7. Duniway, J. M., Hao, J. J., Dopkins, D. M., Ajwa, H., and Browne, G. T. 2001. Chemical, cultural, and biological alternatives to methyl bromide for strawberry. Abstract 41. Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, San Diego, CA. November 5-9.

8. Duniway, J. M., Xiao, C. L., Ajwa, H., and Gubler, W. D. 1999. Chemical and cultural alternatives to methyl bromide fumigation of soil for strawberry. Abstract 3. Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, San Diego, CA. November 1-4.

9. Duniway, J. M., Xiao, C. L., and Gubler, W. D. 1998. Response of strawberry to soil fumigation: microbial mechanisms and some alternatives to methyl bromide. Abstract 6. Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, Orlando, FL. December 7-9.

10. Hao, J. J., Duniway, J. M., Dopkins, D. M., and Xiao, C. L. 2002. Effects of rhizobacteria on inhibition of soilborne pathogens and growth of strawberry. Phytopathology 92:S34 (Abstract).

11. Harman, G. E. 2000. Myths and dogmas of biological control: Changes in perceptions derived from research of Trichoderma harzianum T-22. Plant Disease 84:377-393.

12. Kim, D.-S., Cook, R. J., and Weller, D. M. 1997. Bacillus sp. L324-92 for biological control of three root diseases of wheat grown with reduced tillage. Phytopathology 87:551-558.

13. Kluepfel, D. A. 1993. The behavior and tracking of bacteria in the rhizosphere. Annu. Rev. Phytopathology 31:441-472.

14. Linderman, R. G. 1986. Managing rhizosphere microorganisms in the production of horticultural crops. HortScience 21:1299-1302.

15. Pryor, A. 1999. Results of 2 years of field trials using ozone gas as a soil treatment. Abstract 32. Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, San Diego, CA. November 1-4.

16. Xiao, C. L., and Duniway, J. M. 1998. Bacterial population responses to soil fumigation and their effects on strawberry growth. Phytopathology 88:S100 (Abstract).

17. Xiao, C. L., and Duniway, J. M. 1998. Frequency of isolation and pathogenicity of fungi on roots of strawberry in fumigated and nonfumigated soils. Phytopathology 88:S100 (Abstract).

18. Van Loon, L. C., Bakker, P. A. H. M., and Pieterse, C. M. J. 1998. Systemic resistance induced by rhizosphere bacteria. Annu Rev. Phytopathology 36:453-483.

19. Weller, D. M. 1988. Biological control of soilborne plant pathogens in the rhizosphere with bacteria. Annu. Rev. Phytopathology 26:379-407.

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