Problem: The purpose of this project is to investigate root-microbe interactions to aid development of sustainable management strategies of soil-borne diseases such as apple replant disease (ARD). One of the greatest challenges facing apple production in the Northeast and worldwide is the development of sustainable and economically viable ARD management strategies. Due to the lack of suitable orchard sites, existing sites are continuously planted with apple. This leads to a buildup of soil-borne fungal pathogens such as Pythium, Fusarium, Cylindrocarpon and Rhizoctonia. Pathogenic fungi target the absorptive roots needed for water and nutrient uptake leading to root death, stunted tree growth and tree mortality.
Historically, ARD management strategies have targeted soil-borne pathogens by sterilizing soil with chemical fumigants or biofumigants. In the Northeast, growers commonly manage ARD through crop rotation or biofumigation. However, these management strategies are associated with high economic costs. For example, growers lose income when their land is taken out of fruit production for pre-plant treatments. Growers also make a substantial monetary investment to plant new trees, particularly in high-density apple orchards (1,000 trees per acre). In addition, crop rotations do not always manage ARD effectively. Therefore, if pre-plant strategies are ineffective, establishment and development of young trees may be delayed or trees may die. This results in further loss of income and additional establishment costs. Moreover, while biofumigation effectively suppresses pathogen populations, it also suppresses beneficial microbes.
Sustainable and economically viable management strategies are needed for both conventional and organic apple production to manage ARD. However, there is little understanding of the interactions between apple roots of young trees, beneficial fungi such as arbuscular mycorrhizal fungi (AMF) and pathogenic fungi implicated in ARD. Growers need sustainable and effective management strategies to grow healthy roots and encourage root interactions with AMF. If beneficial microbes can be enhanced, perhaps by the use of organic fertilizers, roots may be less susceptible to pathogen infection at planting and through the life of the orchard.
Justification and Rationale: Traditionally, ARD research focused on identifying associated pathogens, breeding tolerant rootstocks and identifying effective soil fumigation treatments. These research efforts were not designed to determine why certain physiological and environmental factors in a field setting are associated with ARD development. This knowledge gap has led to an increased dependence on fumigation for ARD management and economic loss where pre-plant treatments are ineffective. Sustainable and environmentally friendly strategies are needed to encourage the growth of healthy and efficient roots. These strategies should enhance root interactions with beneficial fungi and inhibit infection of fungal pathogens. Improved management, however, can only be accomplished through greater understanding of root-microbe interactions at the individual root level. For example, in a study of absorptive roots in apple, growth rates of individual young roots influenced the type of fungi colonizing those roots (12). Fast-growing roots were colonized by AMF as soon as three days after emergence. In contrast, slow-growing roots remained uncolonized or were colonized by non-mycorrhizal fungi (NMF) after nine days. In addition, once a root was colonized by AMF or NMF, it was heavily colonized by that fungus alone. This is notable because it suggests that the presence of AMF may inhibit infection of NMF in young, active, apple roots. Only roots older than 25 days were colonized by both AMF and NMF (12).
In an established apple orchard, I investigated the effects of fruiting (fruit vs. no fruit) and localized-nutrient addition of various nitrogen (N) sources (mineral N, organic N, and water) on root growth and colonization by AMF. Results from this study indicated that for fruiting trees, roots growing in mineral N had the highest root length colonized by AMF. In contrast to mineral N addition, roots growing in organic N had the lowest AMF colonization (P=0.05) in fruiting trees. However, results indicated that more AM hyphae grew in soil surrounding roots in the organic N treatment when trees had fruit (P=0.05). This suggests that roots in organic N may use extramatrical hyphae of AMF to help take up nutrients.
The purpose of this project is to investigate the effect of nitrogen source and location at an apple replant field site on root proliferation, and at the individual root level, growth rate, duration of growth and colonization by AMF and replant pathogens. For this study, I will sample young roots (< 21 days old) because they actively respond to environmental factors such as N addition. In fruit crops, roots older than 21 days generally exhibit low activity for nutrient uptake (3, 13 ). I will also use a novel approach of sampling individual roots to investigate possible consequences of N source on roots of apple trees planted at an apple replant site. I will compare roots of two populations, fast- vserus slow-growing roots, to characterize root interactions with AMF and fungal pathogens implicated in ARD.
Objective 1) Determine the effect of nitrogen (N) source and location at an apple replant site on root proliferation in young ‘Golden Delicious’ apple trees on M.26 rootstock. Expected outcome: Trees with localized addition of the readily available N source, urea, will have more root proliferation than trees with the slow-release organic N addition and the control (water) treatment. Trees grown in the grass row middle where perennial grasses were previously grown will have more root proliferation than trees grown in the herbicide strip where mature apple trees were previously grown.
Objective 2) Determine the effect of nitrogen (N) source on growth rate, duration of growth and colonization of individual apple roots by beneficial fungi and pathogenic fungi implicated in apple replant disease (ARD). Expected outcome: Compared to the organic N and control treatments, trees with localized urea addition will have more fast-growing roots that grow for a longer duration. Colonization by both arbuscular mycorrhizal fungi (AMF) and non-mycorrhizal fungi (NMF) will be moderate. Trees with the slow-release organic N addition will have fewer fast-growing roots with greater AMF colonization and minimal NMF infection. Trees with the control treatment will have more slow-growing roots and higher NMF infection.
Objective 3) Determine the effects of location at an apple replant site on growth rate, duration of growth and colonization by AMF and NMF of individual roots. Expected outcome: Trees planted in the herbicide strip where soil-microbial communities developed in association with apple trees will have more slow-growing roots with higher NMF infection and more putative soil pathogens. Trees grown in the grass row middle where apple trees had little influence on development of soil-microbial communities will have more fast-growing roots, higher AMF colonization and fewer putative pathogens.
Objective 4) Determine the effect of root growth rate on root interactions with AMF and NMF. Expected outcome: Fast-growing roots will be rapidly colonized by AMF compared to slow-growing roots which will be uncolonized or colonized by NMF. The presence of AMF in roots will minimize the likelihood of NMF infection in young absorptive roots. These trends are expected across location and nutrient treatment.
Objective 5) Prepare results for academic and extension audiences at academic meetings and multi-state extension events such as Penn State field days and the Mid-Atlantic Fruit and Vegetable Convention. Results will also be disseminated through written extension materials supported by Penn State as described in the outreach plan.
Field site and experimental design. The field site was located at the Russell E. Larson Agricultural Research Center in Rock Springs, Pennsylvania, USA (40.8°N and 77.9°W, elev. 350 m). This site was chosen because apple trees had previously been planted on this site since 1999. On April 3, 2017, 20 root boxes (root observation windows) were installed at the replant site. Ten root boxes were installed in the grass row middle, and 10 root boxes were installed in the herbicide strip (old tree row). Root boxes were constructed as described by Comas & Eissenstat (2000). Root box dimensions are 0.7 x 0.7 x 0.3 m. The experiment used ‘Golden Delicious’ apple trees on M.26 rootstock (Adams County Nursery Inc.; Aspers, PA). On April 27, 2017; 80 apple trees were planted at 0.45 x 6.1 m spacing. Rows were oriented in a north-south direction. Trees were free standing, and the central leader was pruned before leaf out. During bloom, flowers on all trees were removed on May 22, 2017. Soil at the study site was in the Hagerstown series (mesic Typic Hapludalf) with a pH of 7.1 and an organic matter content of 1.7% in the top 0.25 m of the soil profile. Trees were not fertilized at planting.
The experimental design consisted of two replant locations (grass row middle and herbicide strip) and three localized nitrogen (N) patches (inorganic N, organic N, and unfertilized) with 10 replications each. Apple trees were divided into 20 experimental units of four trees each. The two outer trees were used as guard trees, and the two middle trees were treatment trees facing either side of the root box. Root boxes had two viewing windows to allow observation of and access to newly produced roots. One window was on the north side and a second window on the south side of the root box, each facing a different apple tree. Each viewing window was further divided into two smaller windows that were 0.5-m long and 0.3-m deep (4 smaller windows per box). Smaller windows were made of 3-µm thick sheets of clear acetate film (United State Plastic Corporation; Lima, Ohio) to permit root observation and access for root excision. Soil collected during root box installment was sieved with a 1-mm screen and used to fill in gaps between windows and the undisturbed soil. To minimize temperature fluctuations within the root box, 2.5-cm thick removable Styrofoam® was placed against each window when not in use. A removable wooden lid was also placed over each root box after installation to exclude sunlight and rain.
On September 15, 2017, when fine roots first appeared against the observation windows, one of the three N treatments was applied to each window section. For the unfertilized treatment, two viewing window sections were treated per root box. One window section for each side of the root box that faced a tree was treated so that both treatment trees in the experimental unit had an unfertilized control. Twenty ml of water were applied weekly at the soil-acetate film interface. Water was applied across the top of the window at four locations using a pipette. Organic or inorganic N treatments were randomly assigned to the remaining window sections in each root box. For the organic-N treatment, fully expanded green leaves were randomly collected on May 20th from non-experimental mature ‘Golden Delicious’ apple trees on M.26. Mature trees that were planted in 1997 at an adjacent field site at the Russell E. Larson Agricultural Research Center in Rock Springs, Pennsylvania, USA. Leaves were dried at 70˚C for five days in a drying oven and ground using a ball mill. At the time of application, four 5-mm soil cores were taken to a depth of 10 cm along the soil-acetate film interface of the treatment window. For each treatment window, 5 g of ground leaves were divided and funneled down the holes of the four cores, and soil was backfilled over the ground leaves (Fig. S1b). A subsample of the leaves was analyzed for total nutrient content using an elemental analyzer (Vario MAX cube, Elementar; Langenselbold, Germany; Horneck & Miller, 1998). Gradual release into soil of about 20% of total N contained in ground leaf material (2.2% N) was predicted from May to October in 2013 (Moore et al., 2006). For the inorganic-N treatment, 20 ml of urea in water at 70 ppm in water were applied weekly in the third window section to simulate the concentration of N released by ground apple leaves. Urea was applied across the top of the window at four locations using a pipette.
Soil water content and soil temperature were measured weekly at depths of 15 and 30 cm in each root box using a 10-cm long time domain reflectometry waveguide (TDR 100; Campbell Scientific Inc.; Logan, UT) and a HH21 microprocessor-based thermometer (OMEGA Engineering, Inc.; Stamford, CT), respectively. The waveguide and thermometer were inserted horizontally and perpendicular to the treatment windows. Soil solution was collected weekly in the treatment window sections of 10 root boxes using 5-cm-long micro-lysimeters (Rhizon soil moisture samplers, Eijkelkamp Agrisearch Equipment; Giesbeek, The Netherlands). This solution was sampled 24 h after urea application and stored in vials at -20˚C until further analysis.
Root observation and harvest. New root growth was traced every two to three days on the acetate film of the root box windows using a different colored marker (Uchida of America, Corp.; Torrance, California) for each measurement date. Roots were traced from September 13 to October 24, 2017. New roots were also traced on the day of root collection in addition to 1, 2, 4, and 6 days prior to collection from the windows. This approach gave us more detailed information on roots at an early age while the total age ranged from 1 to 70 days old for the first observation period and 1 to 28 days old for the second observation period. On August 14 and October 25, fine roots were harvested and stored in 1.5-ml Eppendorf tubes at 4˚C until further analysis. The acetate film used for the treatment windows was collected from the root boxes.
Future analysis. The acetate film with root tracings will be cleaned and scanned at 400 dots per inch (DPI) using an Epson Perfection 4490 Photo Scanner (Epson America, Inc.; Long Beach, CA). Scanned images will be analyzed for root length, root number, and root age based on tracing color using the software WinRhizo Pro 2007 (Regent Instruments Inc., Canada) and ImageJ 1.46r (Schneider et al., 2012).
A subsample of roots of different ages will be selected for microscopic observations. Selected roots will be cut lengthwise, and half of the root will be used for microscopic quantification of fungal colonization while the other half will be used for DNA sequencing and identification of fungi. Dividing roots for microscopic data and sequencing allows the direct linkage between growth rates of absorptive roots and the identities of specific fungal species colonizing roots in response to N source and replant location.
Roots selected for microscopy will be cleared in 10% KOH at 75˚C and stained with 0.05% trypan blue in a lactic acid:glycerol:water (1:1:1) solution for 30 minutes. Roots will be destained in a lactic acid:glycerol:water (1:1:1) solution overnight and stored in destain solution until observation (Brundrett et al., 1996). Samples will be viewed with a compound light microscope. Individual roots will be mounted parallel to the long axis of the microscope slide with grid lines and covered with a 40×22 mm cover slip. At each grid intersection, fungal colonization will be recorded to determine the percentage of root length colonized. Roots showing signs of colonization by AMF and NMF will be selected for DNA sequencing and fungal identification using next-generation sequencing.
Microbial diversity in soil is generally very high, but the fungi of interest will only include AMF and the small percentage of soil-borne fungi implicated in apple replant disease which are Phytophthora spp., Pythium spp., Cylindrocarpon spp., and Rhizoctonia spp. To investigate which fungal groups colonized the root, 120 root samples with known growth rates will be selected for next-generation DNA sequencing.
Extramatrical and internal fungal development. Soil samples were collected from behind each treated window section in 20-ml scintillation vials. Samples were collected from all root boxes when roots were harvested. Soil samples were stored at -80˚C until further analysis.
Future analysis. Soil samples will be freeze dried at -5˚C for 4 days. Neutral lipid fatty acid (NLFA) and phospholipid fatty acid (PLFA) extractions will used to estimate extramatrical hyphae associated with nutrient foraging and spore abundance of arbuscular mycorrhizal (AM) fungi in the soil behind the treatment windows. Phospholipid and neutral lipid extraction and analysis will follow the method described by Bossio et al. (1998). Briefly, neutral lipids and the polar lipids, containing phospholipids, will be extracted from the soil using organic solvents. The internal standards for PLFA and NLFA will be the fatty acid methyl esters 21:0 and 19:0 respectively (Avanti Polar Lipids; Alabaster, AL). Lipids will be separated into NLFA and PLFA fractions using silicic acid solid-phase chromatography columns. Lastly NLFA and PLFA will be converted into fatty acid methyl esters (FAME) through methanolysis. Extracted FAMEs will be analyzed using a HP GC-FID (HP6890 series, Agilent Technologies, Inc.; Clara, CA) gas chromatograph. External FAME standards (K101 FAME mix, Grace; Columbia, MD) will be used to determine biomass. Biomarkers will be identified using the Sherlock System (v. 6.1, MIDI, Inc.; Newark, DE).
Additional considerations: Trees will be managed with standard agricultural practices for the eastern United States. Available N in soil solution in the N patch will be measured weekly with Rhizon soil moisture samplers (Eijkelkamp Agrisearch Equipment; Giesbeek, The Netherlands). Soil moisture content, soil temperature and shoot length will be measured bi-weekly. Shoot extension and trunk cross-sectional area will be measured to quantify aboveground tree growth in response to replant site location.
Statistical analysis: Data will be analyzed using analysis of variance (ANOVA) or analysis of covariance (ANCOVA) to test for statistical differences between replant location and N source using SAS’s Mixed Procedure (Cary, NC). Data will be transformed when appropriate. Relationships between root growth rate and AMF and replant pathogens will be investigated with regression analysis.
Future research. This experiment will be repeated for the 2018 field season.
Data are still being collected and processed for the 2017 field season.