Determination of the relationship between soil nutrients, mycorrhizae, and plant health in organic blueberry production
Our activities in 2008 for the farm survey portion of the project included:
1) identifying blueberry farms with similar soil types, cultivar, and field age to compare mycorrhizal colonization, soil biology characteristics (enzyme activity, light-fraction organic matter, labile carbon and nitrogen pools) and plant health on matched pairs of organic and conventional Michigan blueberry farms,
2) sampling fruit, leaves, roots, and soil from the farms,
3) developing methods for assessment of mycorrhizal colonization and soil biological characteristics,
4) sample processing and data collection,
5) evaluation and dissemination of first-year results. Significant findings in the farm survey included significantly higher respiration, nitrification, and cultivable bacteria in soils collected from organic farms.
Conventional farms had 40% less anthracnose fruit rot incidence and slightly lower phosphorus content in leaf tissue than their organic counterparts. Marginally significant differences were found for soil chitinase activity, light-fraction soil organic matter content, and mycorrhizal colonization, which were higher in organically managed soils. We are encouraged by our preliminary results of the farm survey and will continue to sample farms on several dates in 2009.
We also made progress in a greenhouse experiment which proposes to evaluate the interaction between mycorrhizal colonization and fertilization practices in blueberries. We attempted two methods to produce blueberries free of mycorrhizal colonization. Irridiated peat proved to be a suitable substrate for producing non-mycorrhizal rooted shoot-tip cuttings taken from one-year-old nursery-grown blueberries. Non-mycorrhizal plants were inoculated with three different strains of ericoid mycorrhizal fungi and fertilized with protein meal, compost, or synthetic fertilizer. Plants were sensitive to excessive salt content in the compost. A major setback occurred when plants were overgrown with fungi while being held in a cooler over the winter.
We will replenish our stock of non-mycorrhizal rooted cuttings in spring 2009 and identify a source of compost with a lower salt content for use in the experiment.
Dissemination of results was limited to informal conversations with growers and presentation at two conferences due to the preliminary nature of our results.
Primary objective: to understand relationship between mycorrhizal colonization, plant health, and soil biology characteristics in Michigan blueberries.
Our first approach is to measure mycorrhizal colonization, plant health, and soil biology characteristics on organic and conventional Michigan blueberry farms. A comparison of organic and conventional farms will provide insights into above- and below-ground changes that occur after the transition from conventional to organic management practices in blueberry production systems. In other cropping systems there is evidence to suggest that mycorrhizae are more abundant on organic compared to conventional farms but this phenomenon has not been studied in Michigan blueberries. An additional goal of the farm survey is to describe the relationship between mycorrhizal colonization and biological characteristics blueberry soils. Earlier studies have found no consistent relationship between mycorrhizal colonization of blueberries to chemical soil measurements.
Our second approach to understand the role of mycorrhizae in blueberries in response to organic and conventional management is to determine the interactions between fertilizer type and mycorrhizal colonization in a greenhouse study. We will assess the contribution of mycorrhizae to growth and nutrient uptake of blueberries supplied with organic or synthetic nitrogen fertilizers. Ericoid mycorrhizal colonization allows blueberries and other ericaceous plants to access nutrients contained in soil organic matter without the need for nutrients to be present in mineral forms. Fertilization with synthetic nitrogen appears to antagonize mycorrhizal colonization of blueberries, but the effect of organic fertilization on mycorrhizal colonization of blueberries is not well understood. Fertilizer treatments in the study include dairy compost, a protein-based commercial organic fertilizer, and ammonium sulfate, which are used by organic or conventional Michigan blueberry growers. Mycorrhizal inocula include three different species of mycorrhizal fungi which were beneficial to blueberry growth or nutrient uptake in previous greenhouse and field studies.
Our first step was to locate eight pairs of organic and conventional fields matched by soil type, field age, and when possible, by cultivar (Table 1). Mr. Dave Trinka, Michigan Blueberry Growers Cooperative, and Dr. Eric Hanson, Michigan State University (MSU) Department of Horticulture, helped with locating farms for the comparison. USDA-NRCS soil survey maps, available at http://websoilsurvey.nrcs.usda.gov/app/, were used to locate soil series within each farm site.
We collected blueberry leaf and fruit samples from the organic and conventional farms on two days in early August 2008. We incubated the blueberry fruit for 10 days at room temperature and 100% relative humidity, a standard method for recovery of fruit-rotting pathogens in the MSU small fruit pathology laboratory. We collected soil and root samples on two separate trips in late September and early October 2008. Each organic-conventional field pair was sampled on the same day. Soil and root samples were processed and placed in storage within five days of collection. Roots were kept in 50% ethanol at 4°C. Soil was passed through a 2-mm sieve and used in soil incubations (see potential nitrogen mineralization and soil respiration below). Subsamples of sieved soil were submitted to a commercial lab for standard chemical soil analyses and frozen at -80°C for use in enzyme activity assays.
After processing the soil and root samples, we focused on developing a method for assessment of mycorrhizal colonization. Blueberry hair roots are very fine, around 1/10 of a millimeter in diameter and usually highly pigmented due to a accumulation of tannins and other polyphenolics. Natural pigments in field-collected roots obscure the view of mycorrhizae, which are present in the epidermal root cells of blueberries and other ericaceous plants. Therefore, roots need to be cleared of pigments before it is possible to assess mycorrhizal colonization. We needed to find the right balance of clearing time, temperature, concentration of potassium hydroxide solution to remove root pigments but not destroy the anatomical integrity of the fine hair roots. We are grateful to Dr. Wei Yang, Oregon State University, and Ms. Kathy Demchak, The Pennsylvania State University, for their willingness to lend expertise in methods for clearing and staining blueberry roots.
In addition to mycorrhizal colonization, we also measured several biological properties of soil, including light-fraction soil organic matter content, potential nitrogen mineralization, basal soil respiration and modeled labile and passive carbon pools, the activity of five soil enzymes involved in soil carbon and nutrient cycling, and the populations of cultivable fungi and bacteria (Table 2). Existing protocols (Robertson et al., 1999) required slight adaptation for use with blueberry soils, which are unlike most soils due to low pH, high organic matter content, and coarse texture. The measurements of soil biological properties were undertaken with the help of Dr. Stuart Grandy, associate professor, Dr. Kyle Wickings, postdoctoral researcher, Mr. Ninh Hmong, M.S. student, and Tracy Beedy, Ph.D student, in the MSU Crop and Soil Science Department and Paligwende Nikiema, Ph.D student in the MSU Department of Forestry.
Statistical analyses were performed with SAS 9.1.3 software (SAS Institute, Cary, NC). PROC GLIMMIX was used for comparison of mean values between organic and conventional farms. Pairs of organic and conventional farms were specified random blocks. PROC CORR was used for determining the relationship between measured variables. Data for biological soil variables were determined at 0-5 cm soil depth, while mycorrhizal and DSE colonization and chemical soil parameters were determined at 0-30 cm soil depth. Muck soils were excluded from the analysis because there were only two replications on muck soils.
For the mycorrhizal inoculation and fertilizer study, we first focused on devising a method to propagate blueberry plants free of mycorrhizal colonization. Non-mycorrhizal plants were needed because we planned to inoculate plants with mycorrhizal fungi. Comparing between mycorrhizal and non-mycorrhizal plants would allow us to determine if there is a synergism between mycorrhizal colonization and organic fertilization, with the hypothesis that organic fertilizers promote mycorrhizal colonization of blueberries and, in turn, mycorrhizae facilitate uptake of nutrients in organic fertilizers by blueberries.
In our first attempt to produce non-mycorrhizal plants, we used surface-sterilized blueberry cuttings that were obtained from a blueberry planting at the Southwest Michigan Research and Experiment Center, Benton Harbor, MI courtesy of Mr. Dave Francis, farm manager. We used sand as a propagation substrate, which was used successfully to obtain non-mycorrhizal blueberries (Yang, 1999). Shoots emerged from the cuttings but rooting was unsuccessful due to excessive heat and sunlight in the greenhouse, despite an overhead mist that was applied at five-minute intervals. Disappointingly, less than 1% of cuttings rooted. We obtained a difference source of propagation material, one-year-old nursery plants from a blueberry nursery, with the rationale that small cuttings would be more amenable to propagation under fluorescent lights. We decided against using sand as a propagation substrate based on our experience with the hardwood cuttings.
Mr. Pete Callow, a research technician in the MSU small fruit breeding laboratory, suggested that peat moss is preferable to sand for propagating blueberries due to its high water-holding capacity. However, Gorman and Starrett (2003) demonstrated that most commercial sources of peat moss contain ericoid mycorrhizal fungi. To avoid confounding the effects of mycorrhizal inoculation by resident ericoid mycorrhizal fungi contained in peat, we needed to eliminate mycorrhizal fungi. Dr. Marvin Pritts, a professor in horticulture at Cornell University, shared his experience with eliminating mycorrhizal fungi from peat. He noted that autoclaving is unsuitable for sterilizing peat due to its detrimental effect on its physical properties and suggested irradiation instead of autoclaving. Previous studies have utilized irradiation to eliminate ericoid mycorrhizal fungi from both peat and soil (Stribley and Read, 1974; Litten et al., 1992), while autoclaving did not completely eliminate ericoid mycorrhizal fungi from soil (Yang et al., 1996).
We learned the MSU Department of Biosystems and Agriculture Engineering department is studying irradiation of food products. We were able to collaborate with Dr. Sanghyup Jeong, a visiting assistant professor in the department, to obtain a sufficient quantity of irradiated peat for use as a propagation substrate. We exposed the peat to 8 KGr radiation (Read, 1983). We then transferred a subsample onto standard laboratory cultivation media to assess the presence of microorganisms before and after irradiation. No fungi or bacteria were obtained from the irradiated peat, while non-irradiated peat harbored bacteria and fungi, and presumably, mycorrhizal fungi.
We stuck 192 shoot-tip cuttings in the sterilized peat substrate and placed them under fluorescent lighting. After 10 weeks, rooted cuttings were transferred to 10 cm3 plastic pots filled with acid-washed autoclaved sand (Yang, 1999). Plants were inoculated with one of three different species of ericoid mycorrhizal fungi obtained from University of Alberta Microfungi Collection and Herbarium. The species of mycorrhizal fungi were selected based on positive plant growth and (or) nutrient uptake in previous greenhouse and field studies (Yang, 1999; Yang et al., 2002). The mycorrhizal inoculation procedure consisted of adding the fungal inoculum, prepared according to Yang (1999), over the surface of the container media.
The fertilizer treatments, compost, a protein-based commercial organic fertilizer, or ammonium sulfate, were applied at a rate equivalent to 20 lbs N per acre on an aerial basis, assuming 20% nitrogen mineralization of the compost and 60% mineralization of the protein fertilizer over the course of the experiment. Plants were grown in a greenhouse under16-hour day lengths with supplemental light provided by sodium halide bulbs. Beginning in mid-November, plants were moved to an unheated portion of the greenhouse and exposed to ambient day lengths to simulate winter hardening. By mid-December the plants were dormant and were transferred to a 40°F cooler and accumulated 1200 chilling hours, which is necessary for emergence of northern highbush blueberry ‘Bluecrop’ from winter dormancy (Muthalif and Rowland, 1994).
Impacts and Contributions/Outcomes
We observed that field-collected organically grown blueberries had a higher incidence of anthracnose fruit rot, caused by the fungus Colletotrichum acutatum, while conventional (non-organic) fruit had higher incidence of Alternaria fruit rot, caused by Alternaria spp. fungi (Table 3). In the leaf tissue nutrient analysis, phosphorus and calcium levels in conventional fields were lower than organic fields (Table 3), while variation in other nutrients was not attributable to management practices (Table 3, additional leaf nutrients not shown). We found mycorrhizal colonization tended to be higher on organic farms relative to their conventional counterparts (Table 3). We also encountered dark septate endophytes (DSE) in roots, but at lower levels than mycorrhizae (Table 3). DSE colonization was not affected by management practices (Table 3).
Soils collected from organic farms had higher rates of soil respiration and populations of cultivable bacteria than soils from conventional farms (Table 3). Light-fraction organic matter and chitinase activity tended to be higher in soils collected from organic farms, but the difference was not quite statistically significant (Table 3). Potential nitrogen mineralization was not affected by management type, but a higher potential nitrification rate was found in soils from organic farms (Table 3). We observed higher soil pH and calcium content in organically managed soils but no effect of management in other chemical soil properties.
Ericoid mycorrhizal colonization was positively related to soil pH but not significantly related to any other soil measurements, including organic matter content, inorganic nitrogen concentration, or enzyme activity (Table 4). Significant linear correlations were observed among several of the soil parameters we measured (Table 4). However, one sampling date may be not be adequate to provide reliable information on the observed relationships. Samples will be collected on several dates in 2009 to determine if the relationships we observed are consistent over time.
About one month after applying the fertilizer treatments, in November, leaf discoloration and marginal necrosis occurred on plants amended with compost, which we later realized was due to excessive salt content in the compost. Unfortunately, after removing plants from the cooler after their chilling requirement was satisfied, extensive fungal growth was observed on the plants and they did not emerge from dormancy.
We surmise the difference in the prevalence of fruit rotting pathogens between management types is due to differences in the efficacy of conventional and organic fungicides. Fungicides were applied in all fields (both organic and conventional) at least once, and in some cases up to 12 times during the growing season. Trials carried out over the years by Dr. Annemiek Schilder, MSU small fruit pathologist and my major professor, have shown synthetic fungicides are consistently more effective than organic alternatives for suppressing anthracnose fruit rot of blueberry, so higher incidence of anthracnose on organic fruit was expected. However, we did not anticipate a slightly higher incidence of Alternaria rot on conventional blueberries. More Alternaria rot on conventional fruit may be a result of Alternaria spp. fungi filling the void left after anthracnose is effectively suppressed with synthetic fungicides. Fungicides targeted against anthracnose are not necessarily effective against Alternaria rot, which supports our explanation of slightly higher Alternaria incidence on conventional fruit. Overall, a very high incidence of anthracnose in organic blueberries suggests that a lack of post-harvest disease control could be a limiting factor in the expansion of organic blueberry production in the Great Lakes region.
We observed lower phosphorus and calcium content in conventionally managed blueberries, while other nutrients were near optimal range for both organic and conventional blueberries. The fact that organic and conventional blueberry plants have similar leaf tissue nutrient concentrations indicates organic growers are meeting the nutritional needs of plants using organic fertilization practices, and nutritional deficiencies are not likely to limit fruit yield in the organic fields we sampled. This is a positive finding because in other cropping systems, growers sometimes experience difficulty in meeting the nutritional requirements of plants after the transition from a conventional to organic production. However, organic blueberry growers collaborating in our study indicate that plants were deficient in some nutrients, especially nitrogen, during the three-year transition period before their fields were certified as organic. Therefore, more research is needed to understand why nitrogen deficiency occurs in the first few years following the conversion to organic management practices in blueberries.
We observed a trend of higher mycorrhizal colonization on organic farms, although the difference between organic and conventional farms was not quite statistically significant. In addition, mycorrhizal colonization varied from 5 to 30 percent among farm sites, but was not related to biological or chemical soil properties with the exception of a positive relationship with soil pH. One explanation for higher mycorrhizal colonization with increasing soil pH is that mycorrhizae may be an effective means for plants to access organic soil nitrogen, given that blueberries colonized by ericoid mycorrhizal fungi are able to utilize nitrogen contained in a variety of organic polymers. Increasing soil acidity (lower pH) is thought to inhibit microbial activity, and presumably, the capacity for nitrogen immobilization by soil microbes. In contrast, immobilization of fertilizer nitrogen may occur to a greater extent as the soil pH nears neutral. Therefore, fertilizer nitrogen may persist in inorganic form at low extremes of soil pH where microbial growth is inhibited. Continued assessment of mycorrhizal colonization in 2009 will be useful to find whether the positive relationship between mycorrhizal colonization and soil pH is spurious or real. Additionally, collecting roots at several points during in 2009 will be valuable in determining if mycorrhizal colonization varies over the course of the growing season. One unexpected observation in our examination of blueberry roots was colonization by DSE. To our knowledge, DSE colonization of cultivated blueberries is only described in one prior study (Goulart et al., 1993). In natural (unmanaged) ecosystems, DSE appear to be ubiquitous root associates with Ericaceae and other plants, but do not usually colonize roots to the same extent as mycorrhizae in temperate climates. According to Smith and Read (2008), DSE are considered “facultative biotrophs” rather than true mycorrhizal fungi, meaning they are found in roots under certain conditions but may be of lesser importance to plants than mycorrhizal fungi. The potential impacts of DSE on their plant hosts are less understood than mycorrhizal symbioses. We will continue to monitor DSE allow with mycorrhizae in 2009.
A higher rate of nitrification in incubated soils collected from organic farms was unexpected because previous studies have attributed high soil nitrification rates in cultivated blueberry fields relative to adjacent forests to synthetic fertilizer applications (Hanson et al., 2002). Nitrate accumulation in blueberry soils is undesirable in terms of both nutrient use efficiency and environmental stewardship. Nitrate is used less efficiently than ammonium by blueberries and is more prone to environmental leaching and runoff than ammonium and dissolved organic nitrogen, which are adsorbed onto negatively charged soil particles and soil organic matter. Higher soil respiration rates, light-fraction organic matter content, and cultivable bacteria activity suggest greater labile carbon availability in soils under organic management. It is known that nitrification in soil is carried out by both heterotrophic (require organic carbon for growth) and autotrophic (obtain carbon from atmospheric CO2) microorganisms, but the relative contribution of heterotrophic and autotrophic nitrifiers in blueberry soils is not known. It is possible that higher nitrification in organic compared to conventionally managed soils is related to higher availability of labile carbon, which may favor increased rates of heterotrophic nitrification. However, nitrification was not correlated to short-term soil respiration rates, light-fraction organic matter content, or carbohydrolase enzyme activity, which are proxy measures for labile soil carbon pools. Significantly higher pH in organically managed soils probably also contributed to higher rates of nitrification in incubated soils. We will repeat the potential nitrogen mineralization assays in 2009 to determine if the higher nitrification observed in organically managed soils 2008 is consistent. We will also monitor nitrate levels in the field on several dates to determine if nitrate production in soil incubations is related to the form of nitrogen observed in the field.
In the first year of the study, we successfully applied techniques for measurement of mycorrhizal colonization of blueberries and several biological attributes of soils. We observed significant differences between organic and conventional blueberry soils in terms of mycorrhizal colonization and several biological soil properties, which provide evidence that organic and conventional blueberry management practices contrast in their effects on the biology of Michigan blueberry soils. Organic and conventional fields have been shown to differ in biological and chemical soil properties in other cropping systems, including tomatoes in California (Drinkwater et al., 1995) and North Carolina (Liu et al., 2007) and in agronomic crops throughout the USA (Wander et al., 1994). In the second year of the study, we will sample fields more intensively to determine if the differences observed between organic and conventionally managed blueberry soils are consistent over the course of one growing season.
Beyond the organic-conventional comparison, we believe we are providing growers with useful information beyond a standard chemical soil analysis. For example, many blueberry growers we spoke with are interested in whether their blueberries are colonized by mycorrhizae, but to the best of our knowledge, no commercial laboratory provides an assessment of mycorrhizal colonization. The organic growers in our study have invested significant resources in soil-building and are interested in knowing whether soil inputs result in some measureable changes in soil function. Their experience has pointed out that chemical soil analyses provide little or no information about the functions of soil organisms. The biological soil measures we applied in this study have been utilized to address ecological questions in natural ecosystems but have not yet seen widespread application in agroecosystems. Therefore, the immediate implications of our results for blueberry production systems in Michigan are not obvious. Biological measures of soil were specified as a need in reports for the NE-SARE project “Ratcheting up commercial organic high-bush blueberry production systems”.
The significant differences observed between organic and conventionally managed blueberry soils in several of the biological parameters we assessed suggests substantial differences in soil functioning due to management practices. One such difference is the activity of N-acetylglucosaminidase, commonly referred to as chitinase. Chitin contains roughly 6% nitrogen and is major component of cell walls of fungi, nematodes, and insects (Leake and Read, 1990; Gortari and Hours, 2008). Chitinase facilitates the breakdown of chitin in soil. Higher chitinase activity in soils may indicate higher biomass and turnover rate of chitin-containing organisms. In natural ecosystems, chitin is a significant reserve of organic nitrogen in soil. Higher chitinase activity in soils under organic management suggests that soil microbes, including ericoid mycorrhizal fungi, acquire more nitrogen in organic form. Differences in chitinase activity may be related to differences in fertilizer and soil amendments used in organic and conventional fields. Therefore, chitinase may be an important measure of nitrogen cycling on organic farms. Although one sample collection date in fall of 2008 limits our conclusions, we believe that our survey of organic and conventional farms will provide useful insights into the functioning of blueberry soils under organic and conventional management.
The results of our mycorrhizal inoculation experiment were less promising. Positive developments include acquisition of mycorrhizal fungi cultures after a lengthy permit process and development of a method for propagating non-mycorrhizal blueberries. Apparently, damp conditions and above-freezing temperatures during cold storage contributed to fungal growth that decimated plants in our experiment. In hindsight, we realize that overwintering outdoors in a protected location, the standard nursery practice, would have prevented the growth of mold while plants met their chilling requirement. Our current source of compost has excessive salt content for use with container-grown blueberries. We will need to find a source of compost with lower salt content. Despite the setbacks, we obtained more nursery-grown blueberry plants and will attempt to carry out the same experiment with non-mycorrhizal rooted shoot-tip cuttings. The results of the experiment will be provided in our final report.
Due to the preliminary nature of our results, dissemination of results was limited to informal conversations with growers and presentation at two conferences, the 2009 MSU Plant Science Graduate Symposium and the 2009 MSU Organic Research Symposium.
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