1. Introduction
Ornamental nursery production is a lucrative segment of the green industry. In 2014, there were more than 8200 nursery producers in the US, and nursery crop sales comprised 31% of all horticultural sales, exceeding
$4.2 billion [
1]. The ability of nursery producers to provide pristine and blemish-free ornamental plants is greatly impacted by plant diseases [
2]. In particular, soilborne diseases are becoming more problematic, as evidenced by the substantial losses to the nursery industry observed in recent years [
3,
4]. Infections caused by
Phytophthora spp. are commonly observed in woody ornamental nursery crops [
5,
6,
7]. These pathogens can live several years in infected plant tissue, plant debris, soil, and water [
5].
Phytophthora species, belonging to the oomycetes, are easily disseminated by water, and can be spread by irrigation, runoff and flood events in a nursery [
5]. One
Phytophthora species of concern to the nursery industry is
Phytophthora nicotianae, which can infect 255 genera in 90 families [
8] and is one of the most devastating soilborne pathogens in the Southeastern United States [
5,
7]. Another soilborne pathogen of concern to nursery producers is
Rhizoctonia solani, which causes diseases in ornamental crops grown in greenhouses, nurseries, and landscapes. The diseases caused by this pathogen include pre- and post-emergence damping-off, stem rot, foliar blight, and web blight [
9].
Rhizoctonia solani attacks seeds below the soil surface a few days after sowing and can kill very young seedlings soon after they emerge from the soil. Management of these soilborne pathogens is critical to maintain healthy nursery stock.
Chemical fungicides have been the most reliable line of defense to prevent losses due to soilborne pathogens [
10]. For over 40 years, soil fumigation with methyl bromide was the standard pre-plant treatment for management of soilborne pathogens in a wide range of agricultural crops [
11]. When methyl bromide was phased out in the early 21st century, metam sodium or sodium N-methyldithiocarbamate fumigants and post-planting fungicides had to be used to manage soilborne diseases [
12]. Concerns regarding resistance development by pathogens due to continuous use of chemical fungicides, environmental drawbacks and the negative effects to successive crops has led to the search for more environmentally sustainable management practices.
Cover crops have been investigated widely in other fields of agriculture for management of plant pathogens, but research on the impact of cover crops in perennial woody ornamental production is lacking. Traditionally, cover crops are planted between seasons and incorporated into the field soil as a green manure [
13]. This management option makes sense in a seasonal cropping system, but in woody ornamental production, the crop can remain in the field for several years. Cover cropping may take place prior to planting or during production in the rows (between plants) and/or row middles (the aisles between the rows). The cover crop can be mowed or lightly disked into the middles, but deep disking and incorporation is not recommended to avoid possible root damage to the main woody ornamental crop. Incorporating cover crops into soil is important because adding organic matter to soil can increase the competitiveness of beneficial, non-pathogenic microbes [
14,
15]. These beneficial microbes can subsequently outcompete soilborne plant pathogens, thereby protecting the main crop from disease [
16,
17,
18]. For example,
Pseudomonas fluorescens (Flügge) has gained attention as a potential biocontrol agent for many soilborne pathogens [
19,
20,
21].
Pseudomonas species are the largest group of plant growth promoting rhizobacteria involved in biocontrol of plant diseases [
22,
23,
24,
25]. Pseudomonads grow rapidly in the rhizosphere, produce a wide variety of growth-promoting substances and adapt to new environments readily, making them well suited as biocontrol agents in agricultural systems [
26,
27,
28,
29].
The main goal of this research was to investigate the impact of cover crops on soilborne disease suppressiveness. The choice of cover crops for a multi-year production cycle was based on research to be able to protect red maple (
Acer rubrum L.) trunks from arthropod oviposition activity [
30,
31,
32]. Soils from field experimental plots with and without cover crops were evaluated before (pre-disked) and after lightly disking (post-disked) the cover crop into the row middles adjacent to the red maple planting. In order to determine the effect of cover crops on soilborne disease suppressiveness, the soil was evaluated for its ability to suppress
P. nicotianae and
R. solani in greenhouse bioassays using red maple and for pseudononad bacterial populations over a two-year production cycle.
2. Materials and Methods
2.1. Field Experimental Design and Layout
A replicated field experiment was established at Moore Nursery in Irving College, TN, USA (35.583889° N, 85.713056° W) (Warren Co.). Two treatments were evaluated in a randomized complete block design. The treatments included: (1) cover crop and (2) bare rows. The bare rows were maintained using pre- and post-emergent herbicides. The pre-emergent herbicide SureGuard
® (flumioxazin 51%, Valent U.S.A. Corp., Walnut Creek, CA, USA) was applied at a rate of 708.8 g product ha
−1 in November 2015, March 2016, August 2016, and April 2017. The post-emergent herbicide Finale
® (glufosinate-ammonium 11.33%, Bayer Environmental Science, Research Triangle Park, NC, USA) with 80–20 (0.5%) surfactant (Ragan and Massey, Inc., Ponchatoula, LA, USA) was applied as a spot treatment to control persistent weeds. Tree rows were spaced 2.1 m apart with 1.8 m within-row spacing between trees, following current recommendations for field planting [
33]. Each 11 by 11 m plot had 25 red maple trees. Each plot was randomly assigned to the treatments and replicated four times.
2.2. Cover Crop Application
Crimson clover (Trifolium incarnatum L.) and winter wheat (Triticum aestivum L.) (Adams-Briscoe Seed Company, Jackson, GA, USA) were seeded on 15 October 2015 using a Herd GT77 Spreader (Herd Seeder Co., Inc., Logansport, IN, USA). Crimson clover was chosen with the expectation to supply nitrogen to wheat so that an additional amount of nitrogenous fertilizer was not needed. Wheat was chosen due to its height to block insect oviposition on the red maple tree trunk. Due to the requirement to protect tree trunks from insect pests, cover crops were sown as close as possible to red maple trees. On 7 September 2016, crimson clover and annual ryegrass (Lolium multiflorum Lamarck) (Adams-Briscoe Seed Company, Jackson, GA, USA) were sown. Annual ryegrass was chosen because it was appropriate for the insect management portion of the project and, unlike wheat, would germinate on contact with soil. Seeding rates were 16.8, 84.2 and 34 kg ha−1 for crimson clover, wheat, and annual ryegrass, respectively. Cover crops were incorporated (disked) lightly (~2.5 cm) into the soil in August 2016 and 2017 using a tractor (John Deere Model 770, John Deere, Moline, IL, USA) and using a 1.2 m wide model disc (Rigsby Manufacturing Co., Walling, TN, USA).
2.3. Soil Moisture and Temperature Measurement
Soil moisture and temperature were measured bi-weekly from March to August in 2016 and monthly from April to August in 2017 (
Figure 1;
Figure 2). Soil temperature was measured with an infrared temperature meter (Spectrum Technologies, Inc., East Plainfield, IL, USA) in probe mode, while soil moisture was measured with a FieldScout time domain reflectometer (TDR) soil moisture meter (Spectrum Technologies, Inc., East Plainfield, IL, USA) using a 7.6 cm probe length inserted to a depth of ~7.0 cm. Soil moisture was measured in volumetric water content percentage (VWC%) and temperature in Celsius (°C). Soil moistures and temperatures were taken adjacent to three trees in each plot in the first, third and fifth rows along a diagonal. Three readings at each tree were taken at three different locations—within the tree row, at the interface of the row and middle, and in the middle between rows.
2.4. Soil Sampling
For greenhouse bioassays to determine soilborne disease suppressiveness, soil samples were collected from cover crop or bare soil treatment both before disking (May 2016 and 2017) and after disking the cover crop into the soil (August 2016 and 2017). Hereafter, the bioassay conducted on soils collected before cover crop incorporation will be referred to as "pre-disked", and soils evaluated after cover crop incorporation will be referred to as "post-disked". Three soil samples were taken from each treatment plot adjacent to the trees evaluated for soil temperature and moisture. Soil was collected from the nearest edge of the tree row where the interface temperature and moisture were evaluated (about 50 cm from the red maple tree to prevent root damage). A 9 kg soil sample was taken from a 30 cm2 area, with 20 cm in depth, at three sampling points per plot, mixed using a spade in a bucket and then transferred into a plastic bag. Spade, bucket, and hands were cleaned with water and then sterilized with 70% ethanol to prevent contamination between samples. The soil was held in an open plastic bag to allow air circulation. Soil was stored for 1 week at 22 °C in a greenhouse at the Tennessee State University Otis L. Floyd Nursery Research Center in McMinnville, TN, USA (35.680480° N, 85.774580° W) (TSUNRC) before use in tests.
2.5. Fungal Culture and Pathogen Inoculum Preparation
Isolate FBG201507 of
P. nicotianae and isolate FBG201508 of
R. solani were obtained from the culture collection of Dr. Fulya Baysal-Gurel at the TSUNRC. The
R. solani specimen was originally isolated from a diseased red maple plant in 2015 and maintained on potato dextrose agar (PDA) medium. The
P. nicotianae specimen was also originally isolated from a diseased red maple plant in 2015 and was maintained on PARH-V8 medium [
34]. Pathogen virulence was confirmed by inoculating red maples with the
R. solani culture or the
P. nicotianae culture and, subsequently, re-isolating the pathogens from the infected roots. For the
P. nicotianae inoculum, the rice grain method, modified after Holmes and Benson [
35], was followed. Briefly, 25 g of long grain rice in 18 mL deionized water was autoclaved twice for 30 min, and three 7 mm sized plugs of
P. nicotianae-colonized V8 juice agar (100 mL of clarified V8 juice (Campbell, Camden, NJ, USA), 15 g of agar (Sigma-Aldrich, St. Louis, MO, USA), and 900 mL of deionized water) were placed in a 250 mL flask and incubated for two weeks at room temperature. The inoculum in the flask was mixed weekly before final use. Soils in square black plastic pots (10 × 10 × 11 cm) were artificially infested by burying three
P. nicotianae-colonized rice grains in the soil at a 5 cm soil depth. For the
R. solani inoculum, an agar slurry was prepared (one Petri dish of a seven-day-old
R. solani culture blended with 1 L of sterilized distilled water), and each pot was drenched with 100 mL of slurry after seeding [
36].
2.6. Red Maple Seed Collection and Planting
Three thousand ‘Franksred’ red maple (Acer rubrum L.) seeds were collected in April 2016 and 2017 and stored in a refrigerator until use. Red maple seeds were sown into the pots filled with soil collected from the field experiment in May 2016 and 2017, and in August 2016 and 2017. Ten red maple seeds were sown in each pot in the 2016 bioassay, while only five seeds were sown in the 2017 bioassay.
2.7. Greenhouse Bioassays
The greenhouse bioassays were conducted at the TSUNRC. The soil sample from each field plot treatment (cover crop or bare row) and replication was divided into individual square black plastic pots (10 × 10 × 11 cm). Those soils were then used as either inoculated (with R. solani or P. nicotianae) or non-inoculated. For each bioassay, twelve single-pot replications per treatment were arranged in a completely randomized design. Overhead irrigation was set up for 1 min twice per day for the whole experimental period. The pre-disked bioassay was initiated in June 2016 and terminated in October 2016, while the post-disked bioassay was initiated in September 2016 and terminated in January 2017 for the first year. Similarly, pre-disked and post-disked bioassays were conducted between June and October 2017 and between September 2017 and January 2018, respectively, for the second year. The greenhouse was maintained at 27 and 21 ± 2 °C, day and night, respectively, with a 14 h day length and 85% relative humidity.
2.7.1. Evaluation of Red Maple Crop Health
After complete germination of maple seeds, stand data were recorded in the greenhouse bioassays. Dead plants were marked with sterilized toothpicks. Toothpicks were inserted into the soil near dead plants, and the total numbers of dead plants were reported for the damping-off percentage. The remaining plants were removed from pots to evaluate root health. Plants were rinsed for 45 min under running tap water and soil attached to the roots was removed. A visual assessment was conducted to identify root rot severity. Seedlings were evaluated for disease severity using a scale of 0%−100% of the total root system affected at the end of the bioassays. After root assessment, ten randomly selected root samples (~1 cm long root tip, four replicates per treatment) were plated on
Rhizoctonia selective medium [
37] and PARPH-V8 selective medium, respectively. Plates were incubated at 25 °C in the dark (VWR incubator, Radnor, PA, USA). The number of root pieces showing
Rhizoctonia growth was counted after 2 days, while
Phytophthora growth was counted after 5 days. The pathogen recovery percentage was calculated by dividing the root pieces showing pathogen growth by the total root pieces plated and multiplying by 100 for each pathogen.
2.7.2. Pseudomonad Colonies
Pseudomonas selective medium (S1 medium) was prepared following the method developed by Gould and his colleagues [
38]. A 1 g soil sample from each treatment pot was transferred into a 15 mL tube (Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 10 mL of sterilized water. Serial dilution up to 10
−6 was performed for each sample, which was then mixed properly using a vortex. After the settlement of soil particles, 100 µL of supernatant was spread-plated using glass beads (3 mm solid glass beads, Walter Stern, Inc., Manorhaven, NY, USA). Plates were incubated at 25 °C for 3 days. The number of colony forming units (CFUs) per gram of soil sample was calculated from the plate counts, the dilution factor and the plated volume.
2.8. Statistical Analysis
Damping-off percentage, disease severity percentage, pathogen recovery percentage, and pseudomonad CFUs were analyzed among cover crop soil and bare soil by year using a generalized linear model (PROC GENMOD) fitted to a normal distribution. The interaction between the soil management strategy (cover crop or bare soil) and disking (pre-disked and post-disked) was analyzed using the same model, and Least Square (LS) means were separated by Tukey’s Multiple Comparison Test at α = 0.05 (SAS 9.4, SAS Institute, Inc., Cary, NC, USA). The pseudomonad CFUs were log transformed for analysis purposes, but original mean values are presented in the figures. All analyses were performed for both pathogen-infested soil and non-infested soil.
4. Discussion
Cover crops are known to prevent soil erosion [
39,
40] and effect weed suppression [
41,
42], and are capable of changing soil microbial populations [
14]. These characteristics are often related to increased soil fertility in agricultural and ornamental nursery production systems [
43,
44]. The possible mechanisms of soilborne pathogen suppression by cover crop introduction may include: (1) the addition of green manure into the soil, which ultimately becomes organic matter, (2) introduction of or increased beneficial microorganisms [
14], (3) reduced dispersal of pathogens [
45,
46,
47], and (4) toxins/chemicals produced by cover crops that are harmful to pathogens [
48]. The few possible mechanisms found in our systems are discussed below.
In a greenhouse bioassay with
Rhizoctonia solani added, while the incorporation of the cover crop had no effect on the reduction of damping-off and
Rhizoctonia root rot disease severity in 2016, the incorporation of cover crops into the soil reduced
Rhizoctonia root rot disease severity in 2017. Our results show that cover crop usage reduced
Rhizoctonia pathogen recovery during the two years of the experiment, suggesting that when
Rhizoctonia root rot disease is severe, cover crop usage can reduce disease severity. Wen and colleagues evaluated a cereal rye cover crop for effectiveness at suppressing
R. solani and found that short-term cereal rye cover cropping lowered the severity of
Rhizoctonia root rot on soybean in
R. solani-inoculated soils [
49]. They also highlighted that a longer period of cover cropping may be required to see the effect of the cover crop on pathogen suppression. These results could be related to the addition of soil organic amendments and mineral nutrients. Baysal-Gurel and her colleagues also found suppression of
R. solani in woody ornamental production by use of cover crops as a source of green manure [
50]. Amending soil with plant residues has been proposed to enhance organic matter and soil fertility and reduce the severity and incidence of diseases caused by soilborne pathogens [
51]. Another reason for soilborne disease suppressiveness with the usage of a cover crop can be soil moisture and/or soil temperature. In our red maple production field, the soil temperature (7 cm below the soil surface) was monitored, and it was found that cover crop treatment had lowered the soil temperature compared to the bare treatment [
32]. Similarly, lower soil moisture was recorded during the cover crop growing period in cover crop soil compared to bare soil. The level of moisture in the soil has been found to be associated with soil microorganism activity in many studies [
52,
53,
54,
55,
56,
57]. Less moisture reduces the opportunity for pathogen development [
52], and this could explain how the higher moisture and temperature in bare soil facilitated pathogen development in our system. The increased concentration of pseudomonads in the soil can explain the effectiveness of cover crop incorporation.
A similar reduction in disease was seen in a greenhouse bioassay with
Phytophthora nicotianae added in the soil. The incorporated cover crop may reduce the opportunity for dissemination of
P. nicotianae spores in soil. Ristaino and Johnston found that wheat or rye cover crop introduction to a bell pepper field reduced the splash dispersal of
Phytophthora pathogens [
46]. Similarly, the bare field treatment had the maximum disease incidence and higher spread across the rows of the bell pepper field. In a no-till system, more diverse soil microorganisms were observed [
58], which could be in the form of soilborne pathogens. The addition of organic amendments to the soil also has been associated with the reduction of soilborne
Phytophthora in many systems [
51,
59,
60,
61,
62]. In our study, cover crop incorporation by lightly disking into the soil was efficient at reducing
Phytophthora root rot disease severity compared to bare soil. Incorporation of the cover crop into the soil, which led to increased organic amendments, reduced moisture and temperature, and caused increased pseudomonad populations, which most likely played a role in
Phytophthora disease suppression.
The introduction of fluorescent pseudomonads has been associated with decreased soilborne pathogen populations in cover crops and other systems [
63,
64,
65,
66,
67]. The main properties of pseudomonads are their ability to grow rapidly in the rhizosphere, their production of a wide variety of growth-promoting substances, and their ability to adapt to new environments. The level of pseudomonad count in our cover crop treated soils increased noticeably in the second year of the production cycle in
R. solani-inoculated soil, which could be due to the two years of cumulative effects from cover crop residues. Interestingly, cover crops were able to enhance beneficial microorganism levels in the soil, which likely had antagonistic activity to suppress soilborne pathogens. Pathogen suppression has also been observed in wheat fields, where the cultivation of wheat cultivars enhanced the production of fluorescent pseudomonads, which had antagonistic activities against
Rhizoctonia root rot of apples and other ornamental pathogens [
68,
69]. Large populations of
Pseudomonas spp. were also found with cover crop use in carrot cultivation [
70] and cover crops of oat and spring vetch in
Scorzonera cultivation [
71].
Suppressive soil can be defined as soil with the capacity to develop a very low level of disease even in the presence of virulent pathogens [
69]. It has been found that both biotic and abiotic components in soil are equally important to suppress pathogen levels. Therefore, properties such as chemical and physical properties, conditions such as organic amendments, microbes such as pseudomonads, and abiotic factors such as temperature and moisture are important attributes to consider for pathogen suppressiveness [
67]. Similarly, making certain manipulations to the soil, such as the introduction of cover crops in the production system, can induce the suppressiveness characteristics. The properties were enhanced either by the presence of a cover crop as a standing crop or as organic amendments added beneath the soil, changing the damping-off, disease severity, pathogen recovery, and soil microbial status in our system. Some of the microbial status changes were more pronounced depending on the type of pathogen. However, the general finding was that cover crops were able to reduce the levels of the two pathogens introduced into our system by inducing disease supressiveness characteristics of the soil.
We found evidence that the cover crop reduced the pathogen pressure in the red maple nursery production system. The incorporation of cover crops into the soil and the associated biotic and abiotic factors were able to increase soilborne disease suppressiveness. The reduced levels of damping-off, disease severity and pathogen recovery in the presence of a cover crop suggest that the use of cover crops can be beneficial in a woody ornamental production system. Cover crops can aid the primary crop by reducing the need for synthetic crop protection materials, such as fungicides. By incorporating these cover crops into production, it is possible to develop strategies for pest and disease management that reduce our reliance on agrochemicals, leave fewer pesticide residues, and prevent pest and pathogen resistance to pesticide chemistries.
Thus, we recommend more long-term studies to evaluate the relationship between population changes in pseudomonads and soil resiliency against R. solani and P. nicotianae in different cover cropping situations.