Determination of the relationship between soil nutrients, mycorrhizae, and plant health in organic blueberry production

Final Report for GNC08-099

Project Type: Graduate Student
Funds awarded in 2008: $9,900.00
Projected End Date: 12/31/2009
Grant Recipient: Michigan State University
Region: North Central
State: Michigan
Graduate Student:
Faculty Advisor:
Dr. Annemiek Schilder
Michigan State University
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Project Information

Summary:

We conducted an observational study to determine the effect of organic and conventional management on plant health and soil biology in blueberry fields in Michigan. Organically grown fruit had a higher incidence of anthracnose rot while conventionally grown fruit had a higher incidence of Alternaria rot. Mycorrhizal colonization levels were significantly higher in organic blueberries. Organic and conventional blueberry production practices differed in their effects on soil processes. Lower midseason levels of inorganic N but higher levels of potentially mineralizable N in organically managed soils provides evidence for greater reliance on soil processes in meeting crop N needs in organically managed blueberry fields. Short- and long-term soil incubations and potential soil enzyme activity assays demonstrated that organic management practices enhance biological activity in soil while conventionally managed soils tend to accumulates in slow-cycling soil organic matter fractions. Microbial N and P acquisition via secretion of N- and P- degrading enzymes diverged according to management, with a shift towards N acquisition in soils on organic farms and P acquisition in soils on conventional farms.

A greenhouse experiment demonstrated that inoculation with mycorrhizal fungi increased shoot growth of plants fertilized with feather meal, while compost enhanced the survival of mycorrhizal fungi. 165 days after compost and feather meal application, the N-supplying capacity of compost was nearly exhausted while feather meal continued to release N. This finding suggests protein fertilizer is more likely than compost to elevate late-season N levels in blueberry fields.

Introduction:

Farm survey

Highbush blueberries (Vaccinium corymbosum L.) are adapted to moist, coarse-textured acidic soils with high organic matter content, and not easily cultivated in soils lacking these characteristics (Ballinger, 1966; Haynes and Swift, 1986). If basic cultural requirements are met, blueberry fields in Michigan remain productive for 40 years or longer (Hanson and Mandujano, 1997). This is noteworthy because there is evidence that long-term additions of fertilizer N in cropping systems may deplete soil organic matter (SOM) and reduce crop yield potential over time (Khan et al., 2007). Recent studies have shown that mineral N inputs intensify the activity of enzymes linked to mineralization of the most labile soil C pools, resulting in declines in biologically active SOM (Sinsabaugh et al., 2002; Keeler et al., 2009). The remarkable longevity of intensively managed blueberry fields in Michigan indicates that unique characteristics of the highbush blueberry and its culture may create feedbacks between management, soil, and plants such that conclusions drawn from studies conducted in annual cropping or unmanaged ecosystems may not be applicable to blueberry fields in Michigan.

The highbush blueberry hosts a unique symbiosis between roots and soil fungi known as ericoid mycorrhizae (ERM). ERM are thought to perform a similar function to root hairs, which facilitate water and nutrient uptake from soil in most plants but are absent from blueberries and other Ericaceae-family plants (Vander Kloet, 1980). Ericaceous hair roots have a diameter of <100 µm, a C:N ratio of 20:1, and median turnover time of 120 days (Valenzuela-Estrada et al., 2008). In the Ericaceae, hair roots constitute 40 to 90% of the total root biomass, and it is common to find 90% of the outer layer of cells of hair roots colonized by ERM fungi in natural ecosystems (Read, 1996; Scagel, 2003). ERM fungi can utilize nitrogen (N) in virtually all forms that are found in soil, including inorganic (nitrate and ammonium) and organic (amino acids, proteins, chitin, and tannin-precipitated protein) compounds (Read et al., 2004). By synthesizing a wide array of enzymes, ERM are able to attack structural components (e.g. cellulose and lignin) of soil organic matter and mobilize biochemically protected nutrients including N and P (Read, 1996). Studies using isotope-labeled compounds demonstrated that organic N uptake by ERM-colonized plants was significantly higher than by non-ERM plants grown in aseptic culture (Sribley and Read, 1974) and unsterilized soil (Stribley and Read, 1980). ERM play a major role in niche partitioning in natural ecosystems by allowing ericaceous plants to utilize organic forms of N in soil and detritus that is less accessible by ectomycorrhizal plants and not utilized by arbuscular or non-mycorrhizal plants (Michelsen et al., 1996). N contained in more recalcitrant organic matter may be accessed by ERM fungi via secretion of peroxide, which reacts with Fe+2 in acidic soils to generate hydroxyl radicals which catalyze non-specific combustion of lignin molecules (Burke and Cairney, 1998). Ericaceous plants are highly dependent on ERM for N acquisition in their native state, where soil conditions typically limit the breakdown of organic matter and accumulation of inorganic N (Read and Perez-Moreno, 2003). Native Vaccinium spp. in a forest understory obtained 86% of N via ERM hyphae (Hobbie and Hobbie, 2006). The symbiosis is not without cost to the plant, as ERM are a sink for up to 20% of net photosynthetic C (Hobbie and Hobbie, 2006).

In an early study of the ERM colonization in response to varying inorganic N concentration, Stribley and Read (1976) showed that ERM colonization was reduced at high concentrations of inorganic N despite prolific superficial growth of fungal mycelium over roots. This suggested that initial establishment of the symbiosis may be regulated to some extent by the plant host. However, studies conducted under field conditions have shown inconsistent responses of ERM colonization levels to soil N enrichment (Johannson, 2000; Yang et al., 2002), which is likely due in part to the complex nature of the soil environment. In a meta-analysis of studies that included N fertilization or enrichment and measures of mycorrhizal colonization, the percentage of root length colonized by mycorrhizae was reduced by an average of 5.8% (Treseder, 2004)

Blueberries are a major crop in Michigan, and the number of organic blueberry producers in the region is increasing steadily (pers. comm., Michigan Department of Agriculture accredited organic certifiers, list available at http://www.michigan.gov/mda/0,1607,7-125-1569_25516-55175–,00.html). However, organic blueberry culture has not been researched as extensively as conventional blueberry production systems in which use of synthetic fertilizers and pesticides is allowed. According to a survey taken at the 2007 Great Lakes Fruit and Vegetable Expo, regional blueberry growers have considered organic production but are reluctant to transition over a major portion of their production area because of anticipated yield declines during the 3-year transition period and uncertainty associated with the conversion to a somewhat unknown production system. In addition, organic fruit and vegetable growers with diversified crops are interested in producing blueberries on a small scale but are limited by a lack of cultural information on organic blueberry production. In 2008, about 60 acres of blueberries were certified organic in Michigan, which amounts to only 0.3% of the total blueberry production area. A few blueberry farms in the region have been managed with organic practices for more than 30 years, but the majority of certified organic blueberry fields in Michigan began the organic transition after 2005. These certified organic blueberry farms provide an opportunity to compare two distinct management systems in terms of soil microorganism communities, soil biological activity, mycorrhizal colonization, and disease incidence. We hypothesize that: 1) blueberries on organic farms are more N-limited and allocate more C below-ground to increase nutrient uptake, resulting in higher ERM colonization, while ERM will be less abundant on conventional farms due to heightened levels of soil N; 2) turnover rates of labile soil C will be higher on conventional farms due to synthetic N inputs, leading to diminished labile soil C compared to organic farms; 3) the turnover rate of lignin as measured by phenol oxidase and peroxidase activity, will be unaffected by management but correlated to ERM infection due to the lignin-degrading ability of ERM fungi, and 4) the N-supplying capacity of soil from conventional farms will be lower than organic farms due to synthetic N-mediated depletion of labile C pools and resultant lower biological activity and nutrient turnover; and 5) the prevalence of diseases will vary between management systems due to contrasts in pesticide efficacy and other factors.

Greenhouse study

Nitrogen fertilizers in use on organic farms are limited to materials derived from natural sources (http://www.ams.usda.gov/AMSv1.0/nop) such as compost and protein meals. Conventional growers satisfy crop N needs with inorganic nitrogen or urea because synthetic N is generally less costly than organic sources of N. Ericaceous plants such as highbush blueberry (Vaccinium corymbosum L.) are better able to utilize organic nitrogen when colonized by ERM fungi (Stribley and Read, 1980; Bawja and Read, 1986; Sokolovski et al., 2002). Scagel (2005) reported that nursery-grown blueberries supplied with organic fertilizers grew more vigorously when inoculated with ERM fungi, but the growth of plants fertilized with synthetic nutrients was superior to those grown with organic fertilizer and less affected by ERM inoculation. However, Scagel (2005) applied synthetic and organic fertilizers at the same rate of total N and did not consider the possibility for different nitrogen release patterns of organic and synthetic fertilizers over time. High levels of inorganic nitrogen may inhibit ERM colonization of ericaceous plants (Stribley and Read, 1976). The question of whether high availability of organic nitrogen inhibits ERM colonization has not been tested. Organic forms of nitrogen such as amino acids stimulate fungal growth when added to soil (Lucas et al., 2007). In Michigan, soil inorganic nitrogen levels are higher in conventional compared to organic blueberry fields when plant nitrogen demand is highest, but leaf tissue nitrogen content does not differ significantly by management type (unpublished data). In addition, ERM colonization in Michigan is, on average, higher in fields under organic management (unpublished data). Taken together, these data suggest ERM may be particularly important in plant uptake of nutrients from organic fertilizers. The objective of this study was to determine the effect of nitrogen fertilizer type on growth and ERM colonization of container-grown blueberries inoculated with three species of ERM fungi.

Project Objectives:

Our primary objective is to understand the relationship between mycorrhizal colonization, plant health, and soil biology in organic and conventional blueberry fields in Michigan.

Cooperators

Click linked name(s) to expand
  • Annemiek Schilder

Research

Materials and methods:
Farm survey

We carried out an observational study to describe characteristics of plant and soil health on commercial organic and conventional blueberry production fields in Michigan. We located a representative sample of certified-organic production fields and matched each with a conventional field. Pairings were based primarily on USDA National Resource Conservation Service (NRCS) soil type, but cultivar and field age were also matched if possible (Table 1). Weed management, fertilization practices, and types of soil amendments applied differed sharply between the organic and conventional fields. Conventional growers most often used herbicides for managing weeds beneath bushes, while organic growers used various combinations of cultivation, mulch, mowing, and organic herbicides. Nitrogen applied on conventional farms was in the form of synthetic fertilizer, while N inputs on organic farms were in the form of various protein-based organic fertilizers (e.g., feather meal, NatureSafe granular organic fertilizer) and composts. Insect pests and diseases on conventional farms were managed with synthetic pesticides (up to 16 sprays per calendar year), while pest management on organic farms included more cultural (e.g., pruning) and physical (e.g., insect trapping) means of pest control and less frequent application of pesticides.

We harvested one pint of ripe berries from 15 bushes by hand in late July and early August of 2008 and 2009, with disposable gloves worn to prevent cross-contamination between field sites. Soil and root samples were collected on 25-26 Sept, 3-Oct and 9-Oct 2008 and on 6-7 Jul, 21-22 Aug, and 9-10 Oct in 2009. Each site occupied approximately 0.5 ha and was sampled according to a stratified sampling plan (Dick et al., 1996). Three 2.5-cm-diameter soil samples were taken beneath the dripline of each of 15 randomly selected blueberry bushes at 0 to 5 cm and 0 to 30 cm depths in 2008 and 0 to 5 cm and 5 to 30 cm depths in 2009 and composited by sampling depth. Large root pieces were collected at 0 to 30 cm depth from the same bushes using a narrow spade. Soil and root samples were placed in insulated coolers alongside ice packs and maintained below 4°C. Samples from each pair of organic and conventional farms were collected on the same day. Soil was passed through a 2-mm sieve within 3 days of collection. Subsamples of sieved, field-moist soil were taken and dried at 80°C for 2 days for determination of gravimetric soil moisture content, stored at 4°C, or stored in a -20°C freezer and stored for up to eight months before processing.

Fifty healthy-looking berries from each site were laid out equidistantly on a raised mesh screen raised above 1 to 2 cm of water in aluminum pans, incubated for 10 days at 100% relative humidity, and then inspected for the presence of the following fruit rot pathogens: Colletotrichum acutatum Simmonds, Alternaria tenuissima (Kunze:Fr) Wiltshire, and Phomopsis vaccinii Shear. The incidence twig blight and cane dieback was recorded on 15 bushes at each field on 25-26 July of 2009.

Soil samples collected at 0- to 30-cm depth in 2008 were air-dried and submitted to a commercial laboratory (A &amp; L Great Lakes Laboratory, Fort Wayne, IN) for analysis of soil organic matter, Bray-1 extractable P, and extractable Ca, Mg, and K using soil test procedures recommended for the North Central Region (http://extension.missouri.edu/explorepdf/specialb/sb1001.pdf). Soil samples collected at 0- to 5-cm and 5- to 30-cm depths in October 2009 were passed through a 2-mm sieve, dried at 80°C for 3 days, ground to a fine powder with a mortar and pestle and analyzed for total C and total N by combustion (NA1500 elemental analyzer (Carlo-Erba, Milan, Italy) (courtesy of Kevin Haynes and Prof. David Rothstein, Soil Biogeochemistry Laboratory, Department of Forestry, Michigan State University). Soil pH of 2009-collected samples was determined on 5 g soil in a 1:1 (w/v) deionized water:soil mixture after shaking for 1 minute and allowing soil particles to settle for 30 minutes (North Central Region Recommended Soil Testing Procedures, http://extension.missouri.edu/explorepdf/specialb/sb1001.pdf). Soil moisture content of 2008- and 2009-collected samples was determined by drying field-moist soil in a drying oven at 80°C for 72 hours.

Light fraction soil organic matter (LF) was isolated from soil by suspending 15 g of air-dried soil in 40 ml sodium polytungstate (NaPT) solution adjusted to a density of 1.7 g per ml in 50-mL centrifuge tubes, shaking for 30 min at 190 oscillations min-1 on an orbital shaker, allowing the heavy fraction to settle overnight, and then vacuum-aspirating the light fraction through a 1-?m glass fiber filter (Millipore, cat. no. APFB04700) (modified from Sollins et al., 1999). After the NaPT was rinsed from the LF with at least 250 ml deionized H2O, the LF was dried at 70°C. After drying, the LF was weighed and then ground with a mortar and pestle for determination of C and N content by combustion as describe above. Light fraction SOM was only determined on sandy soils collected at 0 to 5-cm and 0 to 30-cm depths in 2008.

Labile soil C (Cl) content, along with its decomposition constant (k), and mean residence time (mrt) were determined by measuring CO2 respiration from soil incubated for 461 days. Twenty grams (oven-dry equivalent) of field-moist soil were added to glass vials covered with laboratory film (Parafilm, Chicago, IL). Soil was maintained at 60% water-filled pore space determined gravimetrically (Linn and Doran, 1984). CO2 evolution from soil microcosms was measured twice per week for two months, once every other week up to day 250, and then every four weeks with a LI-820 infrared gas analyzer (Li-Cor Biosciences, Lincoln, NE). Labile C content was estimated according to the equation Rt = (C1 × k)e-kt + c, where Rt is the respiration rate in ?g CO2-C g soil-1 day-1, k is the decomposition constant for the labile C pool, t is the incubation time in days, and c is the rate of CO2 respiration of the slow and recalcitrant SOM pools (McLauchlan and Hobbie, 2004). Modeling of Cl by long-term soil incubation fits an exponential decay function to estimate Cl. The respiration rate was nearly constant for muck soils over the course of the incubation, and similar results have been reported for other soils with high organic matter (Weintraub and Schimel, 2003). Due to the disparity in CO2 release patterns between sand and muck soils, Cl was determined only for sandy soils, and due to the lengthy incubation required, only assessed on soil samples collected in 2008. The amount of C respired over a short-term soil incubation may be a simpler method for determination of labile soil C (McLauchlan and Hobbie, 2004) and was included for comparison with Cl.

Field-moist soil collected in the fall of 2008 at 0- to 5-cm and 0- to 30-cm depths was sieved to 2 mm, added to 125-mL flasks, and adjusted to 60% water holding capacity (Linn and Doran, 1984). Fifteen grams of soil were added to each flask on a dry-weight basis. Flasks were covered with a double layer of laboratory film and incubated at 25°C for 14 days. There were two replicate flasks for each soil sample collected. Inorganic N was extracted in 1M KCl (1:5 m/v). We determined nitrate-N according to Doane and Horwath (2003) and ammonium-N determined by the method of Nelson (1983).

Potential nitrogen mineralization and CO2 respiration of soil were determined in 30-day incubations modified slightly from methods described in Robertson et al. (1999). Field-moist soil was passed through a 2-mm sieve and stored at 4°C for up to 40 days. Ten grams (oven-dry equivalent) of field-moist soil was added to glass vials and adjusted to water holding capacity (WHC) for optimal microbial activity, 55% for sandy soils (Paul et al. 2001) or 65% for muck soils (Zimenko and Revinskaya, 1972). Small vials containing soil were incubated at 25°C within humidity chambers (1-pint jars with water added to 1 cm) and covered with plastic film (Parafilm, Chicago, IL) to allow air exchange but prevent excessive moisture loss. Deionized water was added to vials about once per week to maintain a constant mass throughout the incubation. Inorganic N was extracted from 5 g of air-dry soil in 25 ml of 1M KCl after shaking on an orbital shaker at 125 rpm for 30 minutes. Soil solids were allowed to settle to the bottom of the flask and then the supernatant was poured through filter paper (Whatman No. 2) that was previously leached with 1M KCl. NO3–N and NH4+-N concentrations were determined as described above.

One gram of soil was removed from storage at -20° C and added to 125 ml of 1M sodium acetate buffer at a pH of 5.0. Slurries were continuously mixed on a stir plate while 200-?L aliquots were withdrawn and added to 96-well plastic microplates, followed by 50 ?L of prepared substrates, 4-methylumbelliferone(MUB)-?-D-cellubioside, 4-MUB-?-D-glucoside, 4-MUB-N-acetyl- ?-D-glucosaminide, L-tyrosine-7-amino-4-methylcoumarin, 4-MUB-phosphate, L-dehydroxyphenylalanine, or L-dehydroxyphenylalanine plus 0.3% hydrogen peroxide for determination of activity of the following enzymes: ?-1,4-glucosidase (BG), ?-D-1,4-cellobiosidase (CBH), ?-1,4-N-acetyl-glucosaminidase (NAG), acid phosphatase (PHOS), tyrosine aminopeptidase (TAP), phenol oxidase (POX), and peroxidase activity (PER), respectively. Buffer, buffer plus fluorescent tag, buffer plus substrate, soil slurry plus substrate, and nothing were included to correct for background fluorescence or absorbance. Plates were incubated at 15°C. Incubation times were as follows: PHOS: 2 to 4 hr; BG: 2 to 4 hr; NAG 3 to 4 hr; CBH 4 to 6 hr; PER: 3–5 hr; TAP: 5 to 7 hr, POX: 20–24 hr. Fluorescence (enzymes except PER and POX) or absorbance (POX and PER) was measured on a microplate reader (Thermo Flouroskan and Multiskan, Thermo Scientific, Hudson, NH) and then converted to activity per gram of dry soil per hour (Saiya-Cork et al., 2002).

Cultivable soil microorganisms were enumerated on semi-selective media. Soils were diluted to 10% (w/v) in phosphate-buffered saline (Sanbrook et al., 1989) and placed on a shaker table set at 128 oscillations per minute for 30 minutes. Using field-moist soil that was passed through a 2-mm sieve and stored for up to 14 days at 4°C. The selective media and microbes isolated were Dichloran Rose Bengal Chlortetracycline (DRBC) (King et al., 1979) with chloramphenicol substituted for chlortetracycline for general fungi, STR for Streptomyces spp. (Conn et al. 1998), S1 for fluorescent Pseudomonas spp. (Gould et al., 1985), STR for Streptomyces spp. (Conn et al. 1998), S1 for fluorescent Pseudomonas spp. (Gould et al., 1985), heat treatment at 90°F for 30 minutes and tryptic soy agar (TSA) for Bacillus spp. (Bashan et al., 1993), and 1/10th-strength TSA for general bacteria (Bashan et al., 1993). Trichoderma spp. populations were assessed on Rose Bengal agar in 2008 and on a Trichoderma spp.-selective medium containing several fungicides (Askew and Laing, 1993) in 2009. The volume and soil dilutions plated were, 800 ?l10-1 on TSP, 200 ?l 10-2 on DRBC, STR, S1, and TSA (Bacillus spp.), and 200 ?l 10-4 on 1/10th-strength TSA (bacteria). Beneficial groups of microbes were chosen based on pathogen suppression, plant growth promotion, or induced plant disease resistance attributed to the respective taxa in other studies, for example, total cultivable bacteria (Postma et al., 2008; Bonanomi et al, 2010), Bacillus spp. (Bulluck et al., 2002; Orhan et al., 2006), fluorescent Pseudomonas spp. (Bulluck and Ristaino, 2002; Hass and Defago, 2005; Bonanomi et al., 2009), Streptomyces spp.(Crawford et al., 1993; Aini et al., 2005; Postma et al., 2008), and Trichoderma spp. (Bulluck et al., 2002; Bulluck and Ristaino, 2002; Vargas Gil et al., 2007; Bonanomi et al. 2009), while general media for fungi and bacteria indicate the population size of cultivable species. Drawbacks of the dilution plating method include preferential selection for fungi with prolific sporulation traits (Van Bruggen and Semanov, 2000), disregard for uncultivable species that comprise a sizable and often overlooked component of microbial communities (Torsvik et al., 1990), underrepresentation of taxa with a slow growth response to glucose that are outcompeted by so-called “sugar fungi”, e.g., Mucor spp. and Penicillium spp. (Hudson, 1968; Rice and Currah, 2002), and lack of correspondence between in-situ microbial populations and those assessed on agar media (Thormann, 2006). Sewell (1959) contends that information provided by soil plating methods is not reflective of dominant microflora in acidic and highly organic heath soils, traits shared with blueberry soils in Michigan. Still, the dilution plating method is useful for monitoring changes in microbial communities (Vargas Gil et al., 2007).

Three petri-dish subsamples per soil sample were included in 2008 and this was reduced to two in 2009. After diluted soil was spread on agar, plates were incubated in the dark at 22°C. Colony numbers were assessed after 3 days for fluorescent pseudomonas, general bacteria, and bacilli and after 7 to 10 days for streptomycetes and fungi. If plate counts could not be completed within these intervals they were stored at 4°C in darkness for up to two weeks. Soil moisture content was determined by drying 10 g of soil for 3 days at 80°C. Colony forming units (CFU) per g dry soil was the unit of measure for microbial populations and calculated from the total number of colonies on petri dishes and soil moisture according to the formula

(# of colonies, mean of plate replicates)*(dilution factor)*100
(100 – % soil moisture)

In 2008, fungi, bacteria, and Trichoderma spp. populations were assessed in soil collected at 0- to 5-cm and 0- to 30-cm depths in late September and early October of 2008. In 2009, populations of fungi, bacteria, Trichoderma spp. Bacillus spp., fluorescent Pseudomonas spp., and Streptomyces spp. were assessed in soil samples collected at 0- to 5-cm and 5- to 30-cm depths on 9-10 Oct 2009.

Root pieces up to 30 cm long were washed of adhering soil. Hair roots were carefully excised with a forceps and scalpel blade and placed in 50% ethanol at 4°C for up to 4 months (Stackpoole et al., 2008). Hair roots were soaked for 48 hours after storage in ethanol, in 10% KOH, rinsed three times with distilled water, acidified in 1% aqueous HCl overnight, and stained at room temperature in acidified 0.05% aniline blue (Vohnik et al., 2009). Root segments were then mounted on slides and examined at 600x to 960x magnification on an Olympus IX-71 inverted microscope (Olympus America Inc., Center Valley, PA) equipped with differential interference contrast. Twenty-five 0.5-cm-long by 70-130-?m-diameter root segments were examined per root sample, which equates to about 12 cm of hair roots examined per plant. The method of McGonigle et al. (1990) was modified to assess the number of epidermal cells colonized. The standard gridline-intersect method (Gionovenneti and Mosse, 1980) or root length method (Biermann and Linderman, 1981) was not used due to difficulty in distinguishing between dark septate endophyte (DSE) microsclerotia and ERM at low magnification (10-200x) used for these methods. DSE colonization was recorded as intracellular microsclerotia associated with superficial or intercellular thickened non-staining hyphae (Stoyke and Currah, 1991; Jumpponen and Trappe, 1998). ERM and DSE colonization is expressed as the percentage of epidermal root cells filled with hyphal coils or microsclerotia, respectively (Figure 1). Colonization of individual plants was calculated as the average percentage colonization of hair root cells of 50 (2008) or 25 (2009) root segments. ERM and DSE colonization values in each field site were calculated by averaging the colonization of roots of eight plant replicates for samples collected in fall 2008 and July 2009 and four plants for samples collected in August and October 2009.
Soil dilution plating and colony enumeration on selective media

All statistical analyses were performed in SAS 9.2 (SAS Institute, Cary, NC). Analysis of variance of the fixed effects of management (conventional and organic), soil depth (0 to 5 and 0 to 30 cm in 2008, 0 to 5 cm and 5 to 30 cm in 2009), and sampling date (July, August, and October, determined only in 2009) was carried out in the GLIMMIX procedure. Pairs of conventional and organic farms (n=8) were considered random blocking effects. Six organic-conventional farm pairs were on sandy soils and two pairs were on muck soils. Years were analyzed separately. Because several parameters (Cl, mrt, k, light fraction organic matter) were not measured on muck soils, mucks were omitted from some analyses of 2008 data to allow comparison of organic and conventional management effects across variables. Dependent variables were transformed as needed, e.g., if there was a systematic pattern to the distribution of residuals or if the variances of fixed effects were unequal according to Levene?s test for homogeneity of variance. In addition to inspection of residual plots and Levene?s test, the Box-Cox procedure, which minimizes the residual error sums of squares, was used to determine the optimal transformation for each response variable. Soil type was added as a fixed affect for analysis of ERM and DSE because non-zero estimates were provided for soil type and blocking effects, while these effects were not mutually estimable for analyses of variables (enzyme, microbes, labile C, potential N mineralization) assessed on a bulk-soil basis. PROC CORR was used to determine the relationship between dependent variables measured at 0- to 30-cm depth on sands (n=12) and mucks (n=4); correlation analysis was conducted separately for sand and muck soils because the values of biological measures on muck soils were frequently of an order of magnitude greater than those observed on sands. For 2009 data, responses from samples assessed at 0 to 5 cm and 5 to 30 cm were averaged on a per-unit mass basis according to bulk density values published for each soil type (NRCS soil survey, http://websoilsurvey.nrcs.usda.gov/app/HomePage.htm) prior to correlation analysis, and correlation analyses for sand and muck soils were run separately, as in 2008. SAS PROC FACTOR was used for principal component analysis of biological response variables expressed on a bulk-density-weighted average of samples collected at 0- to 5-cm and 0- to 30-cm depths on sandy soils collected in October 2009. Factor scores were rotated to provide orthogonal contrasts between principal components (Sinsabaugh et al., 2008). Prior to performing the principal component analysis, variables were standardized to a correlation matrix. Anthracnose and Alternaria fruit rot incidence were analyzed as a completely randomized design with management and sampling year as fixed effects because fruit was collected from different field sites in 2008 and 2009 and pairs of organic and conventional farms were not matched by blueberry cultivar in all cases (Table 1).

Greenhouse study

Shoot tip cuttings of Vaccinium corymbosum ‘Bluecrop’ were propagated in a coir substrate (Crop Circles, Lansing, Michigan) that was autoclaved for two hours to eliminate ERM fungi (Vohnik et al., 2003). The coir substrate pH was 5.8 and electrical conductivity (EC) was 0.94 mmhos cm-1, as determined by a commercial soil testing service (A &amp; L Great Lakes Laboratories, Inc.). Rooting of cuttings occurred after 12 to 14 weeks, longer than 6 to 8 weeks normally required for rooting of shoot tip cuttings in peat moss (personal observation). After rooting, plants were transferred to a fine-textured coir substrate (Cocogro, American Agritech, Chandler, AZ) that was mixed with perlite at a ratio of three parts coir to one part perlite by volume. The Cocogro coir had a pH of 6.1, EC of 0.77 mmhos cm-1, and less than 1 ppm inorganic nitrogen. Plants were grown under cool fluorescent bulbs for 28 days and watered with deionized water titrated to a pH of 4.5 with sulfuric acid to maintain the substrate pH in the appropriate pH range for blueberries.
Slant cultures of Oidiodendron maius Barron (UAMH 9263), Rhizoscyphus ericae Zhuang and Korf (UAMH 9270), and an unidentified root-associated fungus of Ericaceae (UAMH 9264) were obtained from the University of Alberta Microfungus Collection and Herbarium (Edmonton, Alberta, Canada). These fungal species were isolated from roots of native Vaccinium angustifolium Ait. or V. corymbosum in Rothrock State Forest, Pennsylvania, USA (Stevens et al., 1997; Yang, 1999). The strains of fungi increased plant growth or nutrient uptake when inoculated onto roots of V. corymbosum in previous greenhouse and field studies (Yang, 1999; Yang et al., 2002). A scalpel blade was used to cut 1 × 1 mm squares of mycelium from the margins of 2-week-old cultures grown on modified Melin Norkrans (MMN) agar (Hutchison, 1981). Two mycelial blocks were transferred to sterile plastic tubes containing 40 ml of liquid MMN medium. Liquid cultures were placed on an orbital shaker and incubated at 23°C under ambient fluorescent light. After 28 days, mycelium was collected on Whatman No. 1 filter paper under partial vacuum pressure and rinsed three times with deionized water. A mycelial suspension was prepared by macerating fungal mycelium in 200 ml deionized water with a Waring 33BL79 blender (Dynamics Corporation, New Hartford, CT) for 90 sec. The final inoculum concentrations were 10 mg ml-1 for O. maius, 13 mg ml-1 for R. ericae, and 17 mg ml-1 for UAMH 9264 on a fresh-weight basis. Slightly different inoculum concentrations were due to different grown rates of fungal species in liquid culture; all mycelium was harvested and used for inoculation. The mixed inoculum treatment consisted of equal volumes of mycelial slurries prepared from each fungal species.

Rooted cuttings were removed from pots, washed of adherent coir under tap water, and roots were placed in 300 ml of mycelial slurry or deionized water (uninoculated control) for 12 hours (Figure 30). A root-dip inoculation method was attempted rather than pipetting mycelial slurries over the growth substrate (Yang, 1999) or beneath plant roots (Jansa and Vosátka, 2000) because the latter two methods resulted in sparse ERM colonization in our preliminary experiments. The viability of the fungal inocula was confirmed by plating 0.1 ml onto potato dextrose agar amended with 10 ppm chloramphenicol on the same day plants were inoculated. The number of colony-forming units (CFU) 10 days after plating was 1, 4, and 2 CFU ml-1 for O. maius, R. ericae, and UAMH 9264, respectively. Following inoculation, plants were transplanted singly into 8.5 × 8.5 × 8.5 cm green plastic containers filled with three parts Cocogro-brand coir to one part perlite by volume.

Five days after inoculation, feather meal and compost fertilizer were incorporated into the upper 2-cm surface of the coir substrate (Figure 31). Separate utensils were used to incorporate fertilizer between inoculation treatments to prevent cross-contamination. The feather meal contained 14.8% nitrogen according to a certificate of analysis provided by the supplier (Morgan’s Composting, Evart, MI). Feather meal was applied at 1.4 g per pot to provide the equivalent of 40 lb of available nitrogen per acre on an aerial basis assuming 65% nitrogen mineralization over the course of the experiment (Hadas and Kautsky, 1994; Gaskell and Smith, 2007). Dairy manure compost (Farm peat, Green Valley Agricultural, Inc., Caledonia, MI) specifically designed for container production (personal communication, Goris Passchier, Green Valley Agricultural, Inc., December 2009) was used in the compost treatment. The dairy compost had a pH of 7.7, EC of 1.75 mmhos cm-1, NO3–N concentration of 79 ppm, nitrogen content of 0.97% and a carbon to nitrogen ratio of 14:1 on a fresh-weight basis (NH4+-N concentration was not determined). The compost was applied at a rate of 30 g per pot assuming 15% nitrogen mineralization (Gaskell and Smith, 2007) to provide a field-rate equivalent of 40 lbs of available nitrogen per acre. Prior to adding the compost, a subsample was titrated with dilute sulfuric acid to estimate the amount of elemental sulfur needed to lower the pH of the compost to 5.0. 1.8 g of powdered sulfur was added to moist compost one month prior use in the experiment.

For the synthetic fertilizer treatment, a solution of 0.3 g ammonium sulfate in 10 ml deionized water was applied and was to be added at the same rate 6 weeks later to provide a split-application to simulate standard fertilization practices for field-grown blueberries (Hanson and Hancock, 1996). However, 21 days after application, the initial ammonium sulfate application was lethal to 38 of 40 plants. In preliminary trials, we applied the same concentration of ammonium sulfate to plants of similar age and stature with no obvious detrimental effects. However, in the preliminary trials, plant roots were not disturbed prior to addition of ammonium sulfate. In the current experiment, adherent coir was rinsed from roots prior to inoculation to facilitate direct contact between fungal inoculum and root tissue. Root disturbance may have increased root sensitivity to high ammonium concentrations. We continued the experiment with the compost and feather meal treatments.

Plastic trays were used for capture of excess leachate from containers, as stipulated by a USDA-APHIS permit that was required for importation of ERM fungi from the UAMH. A nutrient solution without nitrogen (Stribley and Read, 1976) was added to saturate the media 4 weeks after transplanting and every 2 weeks thereafter in both feather meal and compost fertilizer treatments. The nutrient solution was applied because a preliminary experiment showed that plants were deficient in phosphorus after being grown in the Cocogro coir for two months with no supplemental fertilizer. In the first 90 days of the experiment, plants were watered daily with deionized water adjusted to a pH of 4.5 with sulfuric acid. In the latter 90 days, deionized water without added sulfuric acid was used because the pH of the substrate stabilized below 5.5 (Figure 39) in both the compost and feather meal treatments. Daily watering was needed to keep the container substrate moist but not waterlogged. The container substrate was leached with at least 75 ml of water on four occasions over the course of the experiment, the first two instances due to accidental overwatering and intentionally in the latter two instances because the EC of the substrate had reached 2 mmhos cm-1.

Each fertilizer by ERM fungus species combination was replicated eight times in a randomized complete block design. Plant containers in each block were grouped on 40 × 60 cm plastic trays. Over the first 90 days of the experiment, plants were grown in a controlled-environment chamber (Figure 32) at a temperature of 23°C (s.d. ± 1°C) with a 16-hour photoperiod and 95 µmol m-2 s-1 photosynthetically active radiation provided by six 61-cm fluorescent bulbs. Trays were positioned on each of two levels of two growth chambers. The position of trays was rotated within chambers in a systematic pattern every 2 to 3 weeks. After 90 days in the growth chamber, plants were transferred to a greenhouse as the growth chambers were needed for other research projects. The daily minimum and maximum temperatures were monitored on an hourly basis over 5 days. The temperature minima and maxima in the greenhouse were 21°C (± 0.4°C) and 33°C (s.d. ± 4°C). The temperature in root zone of the coir substrate was similar to the ambient air temperature. Plants remained in the greenhouse for the latter 90 days of the experiment.

Shoot growth was measured six times, at the start of the experiment and after new flushes of shoot growth were observed. Shoot length was recorded as the length of plant stems with green leaves. Root length and mass were not determined at destructive plant harvest because it was physically impossible to separate the coir from the fine roots.
The substrate pH and EC were measured five times over the first 50 days of the experiment and 2 weeks prior to the end of the experiment by collecting 10 ml of leachate from 2 randomly sampled pots per fertilizer treatment after flushing with deionized water. The nitrate and ammonium concentration of the leachate was determined at the end of the experiment.

Roots samples were collected for assessment of ERM colonization 50 days after inoculation and at the end of the experiment and stored at 4°C in 50%. Using a scalpel blade, root systems harvested at the end of the experiment were divided in half longitudinally. Each half was sectioned into six clumps; six of the twelve clumps per plant were assessed for colonization by ERM and dark septate endophyte (DSE) microsclerotia. After > 2 mm-diameter roots were removed, roots were placed in histology capsules and cleared in 5% KOH at room temperature for 12 hours. Following the clearing step, roots were soaked in several changes of deionized water until no brown solute leaked from the capsules, soaked in 1% HCl for 2 hours, and then stained at room temperature for 24 hours in 0.05% methyl blue dissolved in a 1:1 mixture of glycerol and water acidified with 1% HCl (modified from Yang, 1999 and Brundrett et al., 1996). After roots were destained in an acidified glycerol-water solution, fine hair roots were excised from large root segments using a scalpel blade, mounted on glass slides, and covered with glass cover slips with slight pressure to flatten cylindrical roots onto a horizontal plane for detailed microscopic examination. ERM or DSE colonization is reported as the number of intracellular hyphal coils (Figure 33) or microsclerotia (Figures 34c and 34d) observed per 50 epidermal cells over an approximate root length of 6 mm (modified from McGonigle et al., 1990). Fifty randomly selected, less than 125 µm-diameter hair roots were examined per plant, corresponding to a total hair root length of 30 cm per plant.

Statistical analysis was performed with SAS 9.2 (SAS Institute, Cary, NC). ERM inoculation and fertilizer effects on ERM and DSE colonization, and fungal inoculum, fertilizer, and date effects on shoot length were evaluated by two- or three-way ANOVA, respectively. Least square mean differences for main and interaction effects were determined using the GLIMMIX procedure with sampling date specified as a repeated factor. The ammonium sulfate fertilizer treatment was omitted from the analysis due to greater than 95% plant mortality. Fifteen percent of plants in the compost and feather meal treatments died over the course of the experiment. Plant mortality did not differ among ERM inoculation or fertilizer treatments.

Research results and discussion:
Farm Survey

In 2009, ERM colonization was higher in organic fields and not significantly affected by sampling date (Table 22, Figure 24). In July 2009, conventional fields had higher soil ammonium + nitrate concentrations, while organic field soils had more potentially mineralizable N (Figures 9 and 10). Soil incubations revealed management-specific effects on labile (active) and slow-cycling (passive) soil C. Labile C content was higher in organically managed soils, while slow-cycling C was more abundant conventionally managed soils (Figures 20 and 6). In October 2009, ammonium + nitrate concentrations were slightly elevated in organic compared to conventional fields (Figure 9), in agreement with previous results from the initial sample collection in fall of 2008 (Figure 7). At 0- to 5-cm soil depth, organically managed soils had higher rates of B-glucosidase (soil C basis, Figure 14) and N-acetylglucosaminidase (soil and soil C basis, Figure 15) activity and a higher ratio of N:P (ratio of N-acetylglucosaminidase + aminopeptidase to acid phosphatase) enzyme activity (soil and soil C basis, Figure 19). Organically grown fruit had nearly 20% higher incidence of anthracnose fruit rot while conventionally grown fruit had 6% higher incidence of Alternaria fruit rot (Figure 2). The Alternaria rot was less prevalent than anthracnose (Figure 2).

Mean values (average of 4 random plant replicates in each field site at three sampling dates) of ERM colonization in sandy soils were positively correlated with soil pH and negatively correlated with soil ammonium, total soil N, beta-glucosidase activity, phosphatase activity, and mean residence time of labile soil C (Table 20, Figure 27). DSE colonization was negatively correlated with phenol oxidase activity and positively correlated with tyrosine aminopeptidase activity (Table 20).

Greenhouse study

Fertilization with ammonium sulfate resulted in plant mortality shortly after it was applied. At the end of the experiment, 180 days after ERM inoculation, an average of 8% hair-root epidermal cells of plants fertilized with compost were colonized by ERM, while roots of plants fertilized with feather meal were nearly devoid of ERM (Figure 36). In the feather meal fertilization treatment, the total shoot length of plants inoculated with the ericoid mycorrhizal (ERM) fungus Rhizoscyphus ericae Zhuang and Korf (UAMH 9270) was significantly greater than plants inoculated with Oidiodendron maius Barron (UAMH 9263), an unidentified ericaceous root-associated fungus (UAMH 9264), or a mixed species inoculum of all three fungi, while the total shoot length of plants inoculated with ERM fungi was greater than uninoculated plants (Figure 37). ERM inoculation did not affect the shoot length of plants fertilized with compost (Table 22 and Figure 37). Plants supplied with compost as an N source had more shoot length than those supplied with feather meal except on the last measurement date (Figure 38); plant shoot length did not increase in the latter half of the experiment due to environmental stress in the greenhouse environment. The pH of the container substrate increased temporarily two weeks after fertilization with feather meal, but not compost amended with elemental sulfur (Figure 39). After 180 days, the concentration of inorganic nitrogen in leachate collected from the container substrate was 25 ppm in the feather meal treatment and less than 1 ppm in the compost treatment (Figure 40), indicating more prolonged nitrogen release from feather meal than compost.

Tables and figures are included in supplemental material presented at the end of this final report.

Participation Summary

Educational & Outreach Activities

Participation Summary

Education/outreach description:
Presentation

“Organic blueberry research at Michigan State University: What we have learned so far.” Michigan’s Organic Agriculture Research Reporting Session. March 6, 2009.

“Soil biology in Michigan blueberries.” Meeting with regional blueberry growers at the Michigan State University Trevor Nichols Research Complex, Fennville, MI. December 18, 2009.

“Effects of conventional and organic management on plant health and soil biology in blueberries.” M.S. defense seminar, Department of Plant Pathology, MSU, April 19, 2010.

“Effects of organic and conventional management on plant health and soil biology in blueberries.” North American Blueberry Research and Extension Meeting, July 27, 2010.

Poster

“Determination of the relationship between soil nutrients, mycorrhizae, and plant health in Michigan blueberries.” MSU Plant Science Graduate Student Symposium, March 25, 2009.

“Effects of organic and conventional management on plant health and soil biology in blueberries.” MSU Department of Plant Pathology Research Symposium, September 18, 2009.

“Effects of organic and conventional management on plant health and soil biology in Michigan blueberries.” Michigan Organic Food &amp; Farming Reporting Session and Poster Contest, March 5, 2010.

“Effects of organic and conventional management on plant health and soil biology in Michigan blueberries.” MSU Plant Science Graduate Student Symposium, March 30, 2010.

“Effects of organic and conventional management on plant health and soil biology in Michigan blueberries.” Organic Farming Conference, La Crosse, WI. February 25-27, 2010.

“Carbon and nutrient cycling and beneficial microorganisms in organic and conventionally managed blueberry soils in Michigan, USA.” Federation of European Microbiological Societies Ecology of Soil Microorganisms Conference, Prague, Czech Republic, April 27-May 1, 2011.

One-on-one meetings with growers participating in the study

Two or three five-minute to two-hour on-farm or telephone visits with each grower-collaborator in 2008 and 2009.

Individualized soil tests for grower collaborators are in preparation with a target for completion in winter 2010-11.

Publications

Sadowsky, J. J., Schilder, A. M. C., and Hanson, E. J. Root colonization by ericoid mycorrhizae and dark septate endophytes in organic and conventional blueberry fields in Michigan. International Journal of Fruit Science, submitted.

-Sadowsky, J. J. 2010. Effects of organic and conventional management on plant health and soil biology in Michigan blueberries. M.S. Thesis, Michigan State University.

-Sadowsky J. J., Schilder, A. M. C., Hanson, E. J., Grandy, A. S., and Hao, J. J. Soil biology in organic and conventionally managed Michigan highbush blueberry fields. In preparation for submission to a peer-reviewed soil science journal.

Project Outcomes

Project outcomes:
Farm survey

Biological activity in soil on organic and conventional farms tended to diverge in patterns of C, N, and P acquisition, as assessed in enzyme activity assays and soil incubations. A brief overview of enzymes involved in soil C, N, and P cycling will aid in interpretation of our findings. Extracellular enzymes that are secreted by soil microbes break down organic matter to simpler compounds that can be assimilated to meet metabolic requirements. Carbohydrolases decompose cellulose and hemicellulose to sugars that are utilized by saprotrophic microbes as a source of energy. Carbohydrolase activity generally increases in response to cellulosic inputs to soil (Sinsabaugh, 2005). In 2009 we assessed the activity of two carbohydrolases: cellobiohydrolase, which cleaves cellobiose from cellulose polymers, and B-glucosidase, which hydrolyzes cellobiose to glucose. N-acquiring enzymes studied in 2009 were N-acetylglucosaminidase, which releases N-acetylglucoasamine from polymers in cell walls of fungi, nematodes, bacteria, and arthropods, and aminopeptidase, which liberates amino acids from peptides and proteins, a refractory but dominant form of N in soil. Acid phosphatase cleaves assimilable phosphate ions from many organic compounds including nucleic acids and phospholipids. In contrast to carbohydrolases, microbial secretion of N- and P-acquiring enzymes depends more on relative demand, such that microbes respond to N or P deficiency by releasing enzymes into the local environment to scavenge for the growth-limiting element (Sinsabaugh et al., 1993).

Higher NAG activity, which measures the release of N-acetylglucosamine from polymers in cell walls of fungi, bacteria, nematodes, and soil arthropods, was observed on organic farms at 0- to 5-cm soil depth in both 2008 and 2009, although the effect was not quite statistically significant. Although NAG was not correlated with inorganic N or potentially mineralizable N, NAG may be important in releasing N for plant uptake in organically managed soils. Tabatabai et al. (2010) observed highly significant correlations between NAG activity and potentially mineralizable N in soils collected from a corn and soy rotation field in Iowa and concluded that NAG is a good indicator of N mineralization in soil.

Another significant finding was that management systems differ in the availability of labile C, as determined by measurement of soil respiration in short and long-term soil incubations, which provides information on the quantity of C available to soil microbes. This effect of management was most pronounced in 0- to 5-cm soil, indicating soil surface C inputs on organic farms may promote microbial activity to a greater extent than those on conventional farms. In addition to the soil respiration data, the notion of greater lability of soil C inputs on organic farms is further supported by significantly higher BG activity, expressed on a SOC basis, at 0- to 5-cm soil depth. Extensive use of herbicides may limit soil organic matter inputs on conventional farms to blueberry litter (both roots and shoots), as opposed to a more diverse array of organic matter on organic farms, including weed biomass, compost, organic fertilizers and sod residues. Blueberry leaves have a C:N ratio near 80:1 (Kourtev et al., 2002) and thus provide a relatively low quality C substrate to saprotrophic soil organisms. Litter inputs of the ericaceous plant Kalmia angustifolia L. tend to suppress microbial activity (Joanisse et al., 2007).

Slow + recalcitrant pool C tended to comprise a more significant portion of total soil C in conventional fields than organic fields. This finding may be related slower breakdown of lignin-C by fungi in response to enrichment with mineral N (Sinsabaugh et al., 2002). As fields are transitioned from conventional to organic, breakdown of this more recalcitrant pool of soil C may resume as mineral N-mediated suppression of lignin decomposition is alleviated. However, the breakdown of high C:N litter likely requires exogenous N for breakdown. N-immobilized as previously accumulated low-quality (high C:N ratio) organic matter is broken down may partially explain the lagging growth and plant N deficiency previously reported by Michigan blueberry growers who have transitioned mature, previously conventional fields to organic management.

Soil nitrogen availability was affected by management practices in fall 2008 and in July and October in 2009. In July 2009, inorganic N availability was higher on conventional farms, while potentially mineralizable N and net nitrification were higher in soils collected from organic farms. Additionally, in fall of both years, organic farms had higher levels of inorganic soil N. The observation of higher potentially mineralizable N in response to organic management was anticipated, as organic fertilizers typically release N over an extended period (Agehara and Warncke, 2005) and the majority of organic growers applied N at the same rate as conventional growers. Likewise, the observation of higher inorganic N availability on conventional farms in July was not surprising because mineral N fertilizers are completely soluble and immediately enrich inorganic N.

Divergence in the quality of C inputs between organic and conventional farms may explain the observed higher rates of nitrification on organic farms. Wurzburger and Hendrick (2007) observed that polyphenolic-rich litter of ericaceous plants may limit nitrate accumulation in soil by suppressing microbial breakdown of organic soil N. There is increasing recognition that heterotrophic bacteria and archaea contribute to nitrification in acidic soils (Prosser and Nicol, 2008). More abundant labile C in soil on organic farms may provide more energy for heterotrophic nitrification.
One of the most striking differences was a highly significant effect of management on the ratio of N:P enzyme activity at 0- to 5-cm soil depth. Analysis of records of blueberry soils submitted by growers the Michigan State University soil testing lab over the years hint that soils are becoming increasingly P-depleted (Hanson, 1989, 2007). Plants are a major sink for soil P (Lindahl et al., 2005) and it is possible that soils of conventionally managed blueberries are relatively P-depleted at shallow depth, especially considering that dense root mats at shallow depth were observed frequently in conventional fields but only in organic fields where dense mulches were employed. It is important to note that prior to the organic transition, organic farms were previously managed with conventional practices. The observed N:P enzyme ratios suggest a shift from a P-limited soil microbial community in conventional fields to one that is more N-limited under organic management.

Currey et al. (2009) reported that N-enrichment suppressed NAG and stimulated PHOS and this scenario may have played out in response to synthetic fertilizer enrichment on conventional blueberry farms. Significant positive relationships between PHOS and N availability have been reported in forest soils (Treseder and Vitousek, 2001). Sinsabaugh et al. (1993) proposed a resource availability model for enzyme activity, such that microbial investment in extracellular enzymes will shift in response to limiting resources, because release of exozymes by microbes is their primary means for acquiring energy and nutrients from the surrounding environment. High N-degrading enzyme activity on organic farms may be important in the breakdown of complex N in organic fertilizers to simpler organic or mineral N available for plant uptake. Reduced N:P enzyme ratios in conventionally managed soils may be related to suppression of N-acquiring enzymes by synthetic N fertilization, high midseason N availability, and relatively higher P demand. Although inorganic N was higher in conventionally managed soils only on the July sampling date, a more pronounced midseason spike in inorganic N may have had a cascading effect on soil enzyme activity over the remainder of the growing season. According to our enzyme assays, N was the most limiting resource in soils on organic farms, while P was more limiting than N in conventional blueberry soils.

Between phenol oxidase and peroxidase, the activity of peroxidase appears to be the main oxidative enzyme in blueberry soils. Sinsabaugh (2010) showed that PER is associated with decomposition of recalcitrant C and is induced by phenolic compounds. For instance, five times higher PER activity was found in biological crusts (surface communities of autotrophic microbes and non-vascular plants) in a burned area compared to an unburned area, and the author attributed the response to reduced supplies of labile C (Sinsabaugh, 2010). It would be interesting to examine whether the peroxidase activity assessed here, with added peroxide to optimize enzyme activity, is of biological origin or instead is a non-biological reaction between added peroxide and soluble Fe (II) ions (Burke and Cairney, 1998). Fe (II) accumulates in acidic soils under waterlogged conditions (McFarlane, 1999). With excess precipitation, fields with shallow water tables are prone to flooding, which was observed in some of the fields sampled in this study. Timonen and Sen (1998) assessed the presence of enzymes in mycorrhizal and non-mycorrhizal roots of Scots pine (Pinus sylvestris L.) grown in non-sterile soil and observed that peroxidase originated primarily from plant roots rather than mycorrhizal fungi, while PHOS enzymes were localized in inter- and extraradical fungal tissue of mycorrhizal roots. This suggests that plant roots may be a significant source of peroxidase activity. Dense mats of roots near the soil surface were observed in some blueberry fields in the current study. To my knowledge, the relative contribution of soil microbes, root-associated fungi, and plant roots to enzyme activity in soil has not been widely studied.

ERM colonization was, overall, about 5% higher on organic farms. This suggests a that a positive effect of organic N on ERM colonization, observed previously in experiments conducted under laboratory conditions (Xiao and Berch, 1999) and with containerized plants (Scagel, 2005; Montalba et al. 2010) may also occur in Michigan blueberries. ERM play a central role in uptake of organic nitrogen by blueberries and other ericaceous plants (Stribley and Read, 1974; Sokolovski et al. 2002; Yang et al.2002; Walker et al. 2010). ERM colonization may aid in direct plant uptake of N supplied by organic fertilizers.

Studies conducted under controlled conditions have suggested that ericoid mycorrhizal (ERM) colonization is reduced as inorganic N availability increases (Stribley and Read, 1976), and contribute relatively less to plant nutrition as N availability increases (Stribley et al., 1975). Conventionally managed soils had higher levels of inorganic N in July, which is a period of peak N demand in blueberries. ERM colonization may compensate to some extent for lower soil inorganic N levels on organic farms than conventional farms that were observed in July by increasing plant utilization of organic soil N, which is at least 50 times more abundant than inorganic N in all soils under study. Determining the levels and composition of dissolved organic N (DON) in soil, whether organic fertilization alters the composition or relative amount of DON to inorganic N, and whether higher levels of ERM colonization facilitate increased uptake of organic N may add to the current understanding soil N cycles in blueberry fields.

A significant management by soil type interaction on ERM colonization indicates that management effects were not consistent across soil types. On the two organic fields with substantially lower ERM colonization than their conventional counterparts on the same soil series, grass sod was maintained in the planting row, while the remainder of organic and conventional fields on sandy soils utilized mulch, cultivation, or herbicides instead of sod culture in the planting rows. In the two organic fields with low ERM colonization levels, grass roots occupied the upper soil profile, and blueberry roots were largely relegated to soil below the sod layer. ERM colonization was reduced at 15-30-cm compared to 0-15 cm soil depth in Oregon blueberry fields (Scagel and Yang, 2005). Extrapolating from these observations provides circumstantial evidence for a negative effect of grass sod on ERM colonization.

Muck soils with > 50% organic matter content did not always support higher ERM colonization levels compared to sandy soil types, indicating that organic matter content alone is a poor predictor of ERM colonization, as reported previously by Goulart et al. (1993). However, the magnitude of the difference in ERM colonization between matched pairs of organic and conventional fields was greatest in muck soils. The reason for this is not known. However, it may be inappropriate to generalize these findings to other blueberry fields planted on muck soils due to the relatively low number of organic and conventional farms on muck soils that were compared in this study.

Highbush blueberries are found in areas with perched water tables (Vander Kloet, 1980) and tolerate transient periods of flooding (Korcak, 1989; Abbott and Gough, 1987b). Wild highbush blueberry stands typically have high levels of ERM colonization (Goulart et al. 1993). ERM fungi may be tolerant of flooding and protect colonized roots from high concentrations of organic acids and other phytotoxic compounds that accumulate under waterlogged, anoxic conditions. Evidence of this was provided by, Leake and Read (1991), who reported that the length of hair roots of the ericaceous plant Calluna vulgaris L. grown in solution at 50 and 100 ppm of acetic acid was significantly reduced in non-ERM plants but remained unaffected in plants inoculated with and colonized by ERM. It is possible that ERM colonization is instead suppressed by excess water in Michigan blueberries due to extended periods of flooding occurring in some fields, which limits root elongation and initiation of new hair roots (Abbott and Gough, 1987b), a prerequisite to initial ERM colonization (Kelley, 1950; Valenzuela-Estrada et al., 2008). Colonization by ERM often lags shortly behind rain events in arid climates (Hutton et al., 1994). ERM in Michigan blueberries should not be limited by soil water availability because most fields are irrigated during dry spells. An inverse relationship between ERM and soil water content was reported by Johansson (2000). We are not aware of any studies that have evaluated the effects of anoxic, waterlogged soils on ERM fungi, or whether ERM colonization protects plant roots under flooded conditions.

ERM colonization was negatively correlated with total soil nitrogen, soil NH4+-N, BG and PHOS activity, and the residence time of labile carbon, and positively correlated to soil pH. Scagel and Yang (2005) also reported that ERM colonization in blueberry production fields in Oregon was negatively correlated with NH4+-N concentration in soil. The inverse relationship between ERM colonization and soil N observed here provides evidence that ERM may be inhibited by high N availability under field conditions, as was previously found in studies carried out under controlled conditions (Stribley and Read, 1976). The observed positive correlation between soil pH and ERM colonization may be related to higher microbiological activity with increasing soil pH. It appears that ERM colonization is especially important for plant uptake of organic N when microbial activity immobilizes mineral forms of N (Walker et al. 2010). Contrary to our results, Stevens et al. (1997) reported that soil pH and ERM colonization in cultivated and native blueberries in Pennsylvania were negatively correlated. ERM colonization of native blueberry plants was not assessed in the current study, and it is possible that intensively managed blueberry soils diverge significantly from those under natural conditions. The negative correlation between PHOS activity and ERM colonization was likewise unexpected, as ERM fungi produce phosphatases which are thought to be important in P nutrition of the host plant (Lemoine et al., 1992). As mentioned above, PHOS activity has been shown to be directly influenced by nitrogen enrichment. Thus, reduced ERM colonization at elevated PHOS levels indirectly may have been a result of high nitrogen availability. The negative correlation between ERM colonization and labile carbon mean residence time is more difficult to interpret than other significant correlations between ERM and soil variables. However, a more lengthy residence time of labile C inputs indicate slower turnover, which may be related to the quality of organic matter inputs entering the soil and the ability of indigenous soil microbial communities to degrade these inputs. It is reasonable to expect that where labile carbon persists for longer periods, biological activity is lower, either because deposited litter is of lower quality and less accessible to microbes, or because the functioning of soil microbes involved in decomposition of labile carbon has been suppressed by some factor unrelated to management practices. Competition between plants and microbes for available soil N will be lower (or less intense) when active carbon pools turn over more slowly. Under these circumstances, ERM colonization would provide less advantage in plant N acquisition because N immobilized by soil microbes as labile C is decomposed would be minimized compared to soils were labile C turns over more rapidly. However, a longer mean residence time of labile C in soil incubations may also indicate more rapid depletion of labile C in situ. We hope that further investigations will be able to unravel the causal factors underlying the significant relationships between the soil parameters and ERM colonization observed in this study.

Less is known about the role of DSE than ERM in Ericaceous plants. Our study confirms that DSE are nearly ubiquitous in Michigan blueberries. DSE occurrence in blueberry roots was significantly affected by soil type and sampling date, with very low root colonization observed on muck soils and higher colonization in July than in August or October. Our results concur with those of Hambleton and Currah (1997), who found that DSE occurred at low levels in roots collected from bog soils. Jumpponen and Trappe (1998) noted that DSE colonization is more abundant on aged, or senescent root tissue and may benefit plants by reducing C demand by degrading non-functional roots or root cells.

The effects of organic and conventional management on populations of cultivable soil microbes were less pronounced than on soil C and N cycling, soil enzyme activity, and ERM colonization levels. This may be due to relatively imprecise estimates of cultivable microbes in comparison to the aforementioned variables because microbes were only assessed in the fall in each year of the study. In 2009 we observed that fluorescent Pseudomonas spp. populations were, in general, enhanced by organic management both at both shallow and deeper soil depths, but highly variable among organic fields. In a study of microorganism populations on organic and conventional tomato farms in Virginia and Maryland, Bulluck et al. (2002) recorded higher populations of cultivable bacteria and Trichoderma spp. on organic farms. However, in a subsequent study in North Carolina, Trichoderma spp. populations were more abundant on conventional farms (Liu et al. 2008). Therefore, it is possible that populations of readily cultivated soil microbes may not reliably responsive to the broad categorization of organic and conventional management practices. Additionally, temporal fluctuations in microbial populations due to variability in soil inputs over the growing season may contribute to high variability in observational studies. It is advisable that future investigations into soil microorganism populations include more rigorous sampling to characterize changes in population sizes over time.

A principal component analysis used to reduce the number of measured variables to two dimensions clearly separated sandy blueberry soils by management practices. The first component represents general carbohydrolase activity while the second component is a contrast between beneficial microbes and PHOS and TAP enzyme activity. A plot of factor scores by management type indicates the soils on organic and conventional farms are more similar at low levels of general biological activity, but at higher biological activity, diverge toward mobilization of N from proteins and increased phosphorus demand (TAP and PER activity) on conventional farms, and more abundant populations of beneficial soil bacteria on organic farms. Interestingly, ERM colonization was only partially correlated with beneficial microbe factor coefficients in principal component two, but was opposed to TAP and PHOS, suggesting factors in soil which contribute to high TAP and PHOS activity (which release amino acids from protein and liberate of phosphate from organic matter, respectively) may be inversely related to ERM colonization.

Lastly, although organic farms tended to have higher populations of beneficial soil bacteria and bacterivorous nematodes, there were also significantly higher populations of root lesion nematodes in soil. Lesion nematodes were detected on roots collected from four out of eight organic farms but not detected in roots collected from conventional farms. The reasons for this finding are not known. It is possible that pathogenic nematodes collected at 0- to 5-cm depth on organic farms were more associated with herbaceous weeds and grasses than blueberry roots, but the observation of lesion nematodes in roots of blueberries may be cause for concern.

Greenhouse study

Results of the greenhouse experiment indicate that plant responses to ERM inoculation depend on the species of ERM fungi well as the source of nitrogen. ERM inoculation increased shoot growth of plants fertilized with feather meal. ERM inoculation did not increase shoot growth of plants fertilized with compost, which indicates that nutrients supplied by compost were equally available to ERM and non-ERM plants. At 165 days after fertilizer treatments were applied, the N-supplying capacity of compost was nearly exhausted while feather meal continued to release N. Therefore, protein-based N fertilizer is more likely than compost to elevate late-summer and fall soil N availability in blueberry fields. High soil N availability in fall may delay tissue hardening and increase the risk of winter injury to plants.

Economic Analysis

Not considered here.

Farmer Adoption

The goal of our study was to determine if organic and conventional management practices differ in their effects on soil biology and plant health. This study characterized many soil variables in blueberry fields that had not been previously investigated, but did not impact cultural practices used blueberry growers over the course of this two-year study. The study also catalyzed further collaboration with organic blueberry growers in the state, including on-farm organic grower-directed studies into organic fertilizers and mulches funded by the Organic Farming Research Foundation in fall 2008 and on-farm studies of compost teas funded by the CERES Trust in fall of 2010.

Recommendations:

Areas needing additional study

Organic N is not typically assessed alongside ammonium and nitrate in crop N budgets but may be a source of N for blueberries. It is not known whether high levels of ericoid mycorrhizal (ERM) colonization under field conditions allow for increased uptake of N forms that are poorly utilized by non-ERM blueberries. Future studies might address the benefit provided by ERM colonization in blueberry fields using labeled isotope tracers to determine if levels of ERM colonization are related to plant uptake of nutrients. In addition, investigation enzyme activities of ERM roots, as was carried out with ectomycorrhizal roots by Courty et al. (2005), may help to elucidate the role of ERM in cultivated blueberries and their response to various management practices. Additionally, it would be of value to match the taxonomic diversity of ERM fungal communities in roots with their functions in soil and plants. Cairney et al. (2000) and Grelet et al. (2005) demonstrated that ericoid fungi show inter- and intraspecific differences in the ability to utilize various forms of organic and inorganic N.

Weed control and especially the effect of mulch thickness and material (wood, straw, and other regionally available materials) on weed pressure need to be researched further. Native blueberries grow in higher organic matter soils with a sequence of an undecomposed surface layer to well-decomposed organic matter which overlies mineral soil. Highbush blueberries often respond positively to mulch, especially on marginal, clay or low organic matter soils and field sites prone to moisture stress (Moore and Pavlis, 1979; Spiers, 1986; Korcak, 1989; Goulart et al., 1997). Based on conversations with growers, the utility of mulch for acceptable weed control is uncertain at best. The effects of mulch depth and width, implications for mulch incorporation into soil for those growers utilizing cultivation when weed pressure becomes high, mulch interaction with fertilizer type (e.g., soluble vs. insoluble organic), trade-offs between mulch depth and N immobilization, and low vigor living mulches such as dwarf clovers, sod, and possibly novel crops adapted to acidic, wet blueberry soils, e.g., wild sorrel need to be investigated further. Lastly, the depth and composition of mulch may interact to affect ERM colonization levels. For example, deeper mulch may immobilize more soluble N and maintain cool soil temperature below the mulch layer, which together would maintain more N in organic form compared to no mulch. Also, under mulch culture, blueberry roots often proliferate in the mulch layer. Researchers generally agree that ERM colonization of ericaceous plants increases uptake of organic N, therefore a testable hypothesis would be that mulched blueberries host higher levels of ERM colonization.

Insect pests are constant bane of organic blueberry growers in Michigan. The most destructive insect pest varied by field site. Some sites are particularly prone to Japanese beetle around harvest and while others were relatively unaffected. Other growers had more problems with fruitworm in spring or blueberry maggot at harvest. The current study did not generate any quantitative data on insect pest prevalence in organic blueberry fields. According to grower conversations, insect damage was most severe on organic farms.

Other important aspects of blueberry culture to investigate are whether it is feasible to cultivate fruit-rot disease susceptible cultivars on organic farms under Michigan conditions and to identify the most appropriate combinations of proactive and reactive treatments for fruit-rot and stem disease control. The highest incidence of fruit rot was observed on organically grown fruit. According to growers collaborating in the study, fruit-rot and stem diseases were a major problem in about half of organic field sites. The most damaging disease varied by site; mummy berry was problematic on sites with cooler springs, stem diseases were most prevalent in older fields, and fruit rot was present to greatest extent on disease-susceptible, early ripening cultivars. Conventional growers sustained lower losses to fruit rot than organic growers, likely due to higher efficacy of synthetic fungicides.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.