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Article

Directing the Apple Rhizobiome toward Resiliency Post-Fumigation

by
Tracey Somera
1,*,
Mark Mazzola
2 and
Chris Cook
3
1
United States Department of Agriculture, Agricultural Research Service, Tree Fruit Research Laboratory, 1104 N. Western Ave, Wenatchee, WA 98801, USA
2
Department of Plant Pathology, Stellenbosch University, Private Bag X1, Matieland 7600, South Africa
3
Tree Fruit Research and Extension Center, Washington State University, 1100 N Western Ave., Wenatchee, WA 98801, USA
*
Author to whom correspondence should be addressed.
Agriculture 2023, 13(11), 2104; https://doi.org/10.3390/agriculture13112104
Submission received: 8 September 2023 / Revised: 21 October 2023 / Accepted: 25 October 2023 / Published: 6 November 2023
(This article belongs to the Special Issue Plant–Soil–Microbe Interactions for Sustainable Crop Production)
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Abstract

:
Currently, there are no standard management practices to counteract the adverse effects of fumigation on the soil microbiome. In this study, a variety of pre-plant soil amendments were examined for their ability to recruit and maintain apple rhizosphere microbiomes that are suppressive to pathogen re-infestation of fumigated orchard soils. The capacity of these amendments to improve other characteristics of soil productivity was also evaluated. Results suggest that composted chicken manure and liquid chitin are likely to be detrimental to plant and soil health when used as a post-fumigation soil amendment. In comparison, insect frass (IF) resulted in a significant increase in tree trunk diameter relative to the fumigated control. Following pathogen re-infestation of fumigated soil, however, IF induced a significant increase in Pythium ultimum in the rhizosphere. Therefore, IF can benefit the growth of young apple trees in fumigated soil but may stimulate pathogen activity upon re-infestation. To date, the possibility of using soil amendments to suppress pathogen re-infestation of fumigated soils has not been tested. Results from this study ground support the use of soil amendments as an intervention strategy for “steering” the soil and rhizosphere microbiome in more beneficial and/or prophylactic directions following fumigation.

1. Introduction

As an apple replant disease (ARD) control strategy, pre-plant soil fumigation is the industry standard. Although fumigation significantly reduces pathogen activity and improves tree growth, this benefit is limited to approximately one year. Post-fumigation, orchard soil rapidly re-establishes a microbial community that is indistinguishable from that found in the corresponding unfumigated replant soil (i.e., a chronic disease state). After one year, multiple soilborne pathogens including the root lesion nematode Pratylenchus penetrans and Pythium species rapidly reinvade fumigated soil and are commonly found at populations higher than that observed prior to treatment [1,2]. Replanting sites where resident soil microbial communities have been impaired by fumigation is particularly well-suited for testing the impact of soil amendments on orchard soil restoration. Currently, there are no standard management practices to counteract the adverse effects of fumigation on the soil microbiome. Mechanical incorporation of pre-plant soil amendments to tree rows following fumigation should fit easily into an orchard management program. Therefore, the application of soil amendments following fumigation is an opportune and crucial time to improve the ability of soil to defend against pathogen re-infestation and improve orchard productivity for an extended period. To that end, our work assessed the potential of a variety of easily attainable and cost-effective amendments to improve biotic and abiotic soil health characteristics following fumigation.
To date, the possibility of using soil amendments, including mustard seed meal, to suppress pathogen re-infestation of fumigated soils has not been tested. When used as an alternative to fumigation, defined Brassicaceous (mustard) seed meal (SM) formulations can provide levels of disease control equivalent or superior to that of fumigation and heightened levels of growth and yield during the initial three growing seasons [1,2]. Multi-year disease suppression occurs because orchard soils treated with Brassicaceous SM sustain elevated populations of specific microorganisms known to suppress plant-pathogenic fungi, oomycetes, and nematodes.
In addition to mustard seed meal, the application of a variety of other types of organic matter (e.g., compost, humus, and charcoal) has been reported to improve tree growth in replant-conducive soil (relative to untreated controls) [3,4]. The application of organic amendments, such as compost, is an accepted management practice to improve soil nutrients, organic matter, and physical composition. Most of the supporting research, however, has evaluated treatment effects over short-term periods (rather than multi-year field trials) and merely assessed plant performance. Assessments of pathogen suppression and resulting disease control are generally not included in these types of studies. Chitin, which makes up between 1 and 15% of fungal cell walls, has also been promoted as a valuable soil amendment with the ability to stimulate potentially beneficial fungal biocontrol agents and control plant parasitic nematodes [5]. However, limited research exists on the ability of chitin-based amendments to control soilborne pathogens of apples. Historically, analyses of rhizosphere and root microbiota have been lacking. Although positive growth results have been consistently linked to disease control, they may be associated with other soil factors such as nutrient availability and organic matter content [6]. Some studies have directly linked compost application to the prolonged suppression of root lesion nematodes [7] but this represents only a single component of the ARD complex. Thus, the biggest gap in previous amendment-based studies has been the failure to link the positive effects of soil amendments with a comprehensive evaluation of the disease system.
A recent study identified the plant rhizosphere as one of the most biodiverse environmental niches on Earth [8]. Over the course of the last decade, it has become clear that the collective of bacteria and fungi living in the rhizosphere is critical for conferring host protection against invading pathogens. It has also become clear that the indigenous microbiome can be engineered for a number of other benefits including drought tolerance [9], nutrient acquisition [10], and modulation of host plant immunity [11]. The primary aim of our study was to explore the potential use of pre-plant soil amendments to enhance long-term microbiome-mediated control of apple replant disease (ARD) following fumigation. At present, there are numerous approaches and conflicting data with respect to the use of organic amendments as a practice to control soilborne pathogens. An adoptable and efficacious management strategy for improving the reproducibility of amendment effects on microbial composition and function requires the development of consistent product composition. Therefore, only materials representing clearly defined organic waste products which are compatible with conventional or organic systems were considered for use in this study. Cost and availability were also factors in selection (Table 1).
These treatments included two different rates (2.2 and 4.4 t ha−1) of a 1:1 formulation of Brassica juncea + Sinapis alba (BjSa) SM, B. napus SM (4.4 t ha−1), a compost derived from the cultivation of shiitake mushrooms, composted chicken-manure, insect frass, and a commercially available product derived from crab and shrimp shells (Table 2).
In addition, Brassica seed meal soil amendments have been shown to reduce several potential postharvest pathogens in soil, including Alternaria and Penicillium [2]. Chitin-based materials have also been shown to elicit plant resistance against fungal postharvest pathogens in apples [12]. This important aspect of orchard health remains relatively unexplored. Thus, a secondary aim of this study was to determine the role of select amendment-modified soil microbial communities in reducing potential postharvest pathogens in the bulk soil.

2. Materials and Methods

2.1. Site Description

All soil used in this study was collected from the Washington State University Sunrise Research Orchard (SRO), Rock Island, WA (latitude 47°18′37″ N, longitude 120°04′01″ W). This location is known to possess the consortia of soilborne pathogens defined as causal agents of apple replant disease [1,13]. The soil type at SRO is Pogue fine sandy loam (46% sand, 5% clay, and 49% silt) with a pH of 6.9 which is close to the optimum pH for tree fruit (6–6.5) [14]. The organic matter content is low (1.2–1.6%) and the nitrogen status of the soil has been described as deficient with NH4-N levels ranging from 1.6 to 3.5 ppm [15] (Soiltest Farm Consultants, Moses Lake, WA, USA).

2.2. Collection and Preparation of Soil

SRO soil was fumigated on 1 April 2021. The old orchard block (14b), previously planted to apple, was removed in 2017. Telone II (1,3-dichloropropene) was applied at a rate of 136 kg ha−1 and injected at a depth of 46 cm. Sectagon K-54 (metam potassium) was also applied at a rate of 356 kg ha−1 and injected into the top 15 cm. Fumigated soil was collected on 26 April 2021. Fumigated soil was collected from the top 30 cm of soil from 20 randomly distributed sites within the central plot area. In addition, approximately 115 liters of unfumigated replant soil were collected from a nearby block containing apple trees (SRO block 12b). Organic residue on the soil surface within tree rows was removed using a shovel; soil was then carefully collected from the top 30 cm so as not to damage existing tree roots. Lids were placed on all buckets to minimize moisture loss and soil was transported back to the lab. Soil was then stored in 115 L bins with lids in a cool and dry location until use. One month after collection, the soil was mixed thoroughly in a cement mixer. The cement mixture was cleaned between soil treatments (fumigated vs. unfumigated, replant-conducive soil) by sterilizing with 2% bleach and rinsing with water three times. Mixed soil was then used in all plant assays.

2.3. ARD Bioassay

This experiment was conducted to confirm disease control in the fumigated soil. Surface-sterilized Gala apple seedlings, germinated and prepared as previously described [16], were grown in pasteurized potting soil (SunGro Sunshine Potting mix #1; Sungrow, Agawam, MA, USA) for 5 weeks. Soil pasteurization was performed by heating overnight at 80 °C on two successive days with 12 h in between each heating session. Seedlings of a similar size were then carefully transplanted into plastic cone-tainers (6.35 cm in diameter, 25.4 cm deep) containing either the fumigated orchard soil, the unfumigated replant-conducive soil, or pasteurized replant soil and placed in a standing rack in a completely randomized design with 10 plants per treatment. Plants were grown in the greenhouse for 4 weeks. Upon harvest, the total seedling biomass, shoot biomass, root biomass, and P. penetrans abundance in fine root tissue were measured as previously described [17].

2.4. Evaluation of Composted Materials

Prior to use in experiments such as soil amendments, the quality, stability, and maturity of both shiitake mushroom compost (derived from mushrooms cultivated on 20% grain/80% hardwood sawdust) and composted chicken manure (non-pelleted, broiler based, and OMRI listed) was evaluated by an approved compost testing laboratory (Soiltest Farm Consultants). The analyses conducted included the moisture and solid content, pH, electrical conductivity (EC), total N, organic C, organic matter, ash, ammonium-N, nitrate-N, C:N ratio, and CO2 evolution (Table S1).

2.5. Soil Amendments

Composted chicken manure (CCM) (non-pelleted). This is an extremely low cost material that is already in widespread use by commercial growers as a fertilizer.
Chitin-based soil amendments: shiitake mushroom compost (SMC), liquid chitin (LC), and insect frass (IF).
Shiitake mushroom compost (SMC). This material was derived from mushrooms cultivated on 20% grain and 80% hardwood sawdust and represents a consistent and commercially available material suitable for organic or conventional production systems, with the potential to be supplied in large volume. It should also be noted that, in this experiment, “fresh” heat-treated SMC, which is almost completely dominated by shiitake mycelium, was selected over “aged” material because it contained a high level of chitin and is more consistent between batches at earlier stages. In addition, this amendment was expected to improve important soil-health characteristics including the water-holding capacity, organic matter, and soil nutrients (1–2% N, 0.2–0.3 P, and 1–2% K).
Liquid chitin (LC). Chitin is a major constituent of crustacean exoskeletons. This product is derived from crab, fish, and shrimp concentrate (SeaShield™, Advancing Eco Agriculture, Middlefield, OH, USA) and represents a consistent and commercially available material which is highly cost-effective (10 USD per gallon).
Insect frass (IF). Insect frass is simply the feces (or castings) of the insect. In this study, mealworm frass was used [18]. This material also contains chitin, which is part of the gut membrane of insects and is present in their frass [19].
Seed meal (SM). The seed meals utilized in this study were derived from Brassica napus cv. Athena [20], Brassica juncea cv. Pacific Gold [21], and Sinapis alba cv. Ida Gold [22]. Seed meal formulations used in all experiments (B. juncea + S. alba and B. napus) were ground, passed through a 1 mm2 sieve, weighed out, and portioned into individual bags for addition to fumigated or unfumigated SRO soil according to the application rates listed in Table 2 (10 or 20 g SM per 2 kg of soil = 2.2 or 4.4 t ha−1). The B. juncea + S. alba SM formulation was prepared by blending B. juncea and S. alba at a ratio of 1:1. Pre-weighed packets of seed meal were thoroughly incorporated into the soil by hand. In total, 2.5 L of seed meal-amended soil was then placed into each pot, moistened with 300 mL autoclaved water, and sealed in gas-impermeable Bitran bags to retain seed meal-generated volatile compounds. The bags were removed after 1 week and pots were maintained in the greenhouse for an additional 6 weeks prior to planting to allow for degradation of potentially phytotoxic compounds.
All soil amendments, with the exception of CCM, were thoroughly hand-mixed into the fumigated soil 2 weeks prior to planting according to the application rates listed in Table 2. The amount of material added to each pot was calculated according to an “applied” volume of 432 m3 of soil per hectare (6340 ft3 per acre) based on a high-density orchard system with a layout of 3.65 m between tree rows, 1.07 m wide weed-free strips, and tilling 15.24 cm deep. The amendment rate for the composted chicken manure treatment was determined as described below.
Amendment rate for composted chicken manure: The EC value of the CCM was assessed (SoilTest Farm Consultants, Moses Lake, WA, USA) and the amount of compost that could be added to 2.5 L SRO soil or 3500 g dry weight without exceeding a soil EC threshold value of 1.7 mmho/cm was calculated [14]. It was predicted that 250 g dry compost would give a final EC value of 1.6 mmho/cm. After adjusting for compost moisture (29%), 322.5 g of “wet” compost was identified as a suitable “per pot” amendment rate (1.7 ton ha−1). The amount of available N in 250 g dry compost was then determined based on soil test results (SoilTest Farm Consultants). Additional nitrogen was applied in the form of ammonium sulfate (21-0-0; N-P-K) to provide 15 g total N per tree as recommended [23]. To avoid potential phytotoxicity resulting from the CCM, rootstock planting occurred 6 weeks after the amendment had been incorporated into the soil.

2.6. Rootstock Genotype

Trials employed the G.11 apple rootstock which reportedly exhibits tolerance to ARD [24].

2.7. Assessment of Rootstock Growth

Upon planting, the initial root volume (mL), trunk diameter (mm; measured at 16–18 cm above soil line), and total rootstock weight (g) were recorded as previously described [17]. For each pot, soil was decanted into a sterilized grey bin and hand-mixed to break up hardened clumps and homogenize any bio-crusts that had formed prior to planting. Bulk soil samples were collected and the remaining soil was placed back into the pot with a single rootstock. After planting, all pots were watered to fill in air spaces around roots. Pots were set up in a completely randomized block design consisting of 7 replicates per treatment (with 7 pots per block) and maintained under standard light and temperature regimes [17]. At the end of the experiment (2 months post-planting), the effect of soil treatments on rootstock growth was assessed by measuring increases in root volume, trunk diameter, and total rootstock weight. The terminal leader-shoot length (cm) was also measured at harvest.

2.8. 16S/ITS Sequencing for Assessment of Microbial Community Composition

In the first experiment in which we attempted to “steer” microbial community composition toward (or away from) different target states when starting from the same environmental conditions, rhizosphere soil was collected from all treatments 4 weeks post-planting and at harvest (8 weeks post-planting). This was conducted by shaking off loosely adhering bulk soil from tree roots and removing firmly attached soil using sterile tweezers. DNA was extracted according to the manufacturer’s instructions from 0.25 g of rhizosphere soil per plant using the DNeasy PowerSoil Kit (Qiagen, Valencia, CA, USA). DNA was then used for bacterial 16S rRNA and fungal ITS amplicon sequencing. For all soil samples, bacterial 16S rRNA and fungal ITS amplicon sequencing were performed by molecular research (Shallowater, TX, USA) as previously described [17]. The Bray–Curtis dissimilarity metric was calculated among treatment groups and used to perform principal coordinates analysis (PCoA) and 1-way analysis of similarity (ANOSIM) of relative abundance data. PCoA was used to visualize movement through community space [25]. Control treatments included (1) fumigated-only control soil, (2) unfumigated replant-conducive soil from the same location, and (3) unfumigated replant soil + BjSa SM (4.4 t ha−1). Since the apple rhizosphere microbiome in fumigated soil rapidly reverts to that of the untreated control, the microbiomes of both untreated orchard replant and fumigated-only soil represent community configurations which are disease-conducive (i.e., compositional arrangements to steer away from). Conversely, the BjSa SM (4.4 t ha−1) control treatment represents a convenient target for assessing the ability of the different soil amendments to direct the community in a positive direction. The ability of this specific seed meal formulation to suppress fungal and nematode pathogens and consistently select a pathogen-suppressive microbiome was demonstrated across multiple orchard locations with different soil characteristics [1,2,17,26]. Two-part tests were conducted in Explicet v. 2.10.5 to identify bacterial and fungal taxa that were significantly enriched (p < 0.05) in select treatment groups relative to the untreated fumigated control.

2.9. Amendment Impacts on Soil Health Properties

As part of the microbial “steering” experiment (i.e., the first experiment), a variety of chemical and physical properties were measured to assess the influence of the soil amendments on overall soil health. The measured properties included the pH, water holding capacity, electrical conductivity, organic matter content, C:N ratio, NO3 and NH4 availability, and total N, P, K (%), Ca, Mg, Zn, B, Cu (ppm) (Soiltest Farm Consultants). These metrics were obtained for bulk soil collected pre-amendment and compared with that of bulk soil collected post-amendment (pooled from all replicate pots within each treatment at harvest).

2.10. (Artificial) Re-Infestation Experiment

Subsequent to the investigations conducted in the microbial steering experiment (i.e., the first experiment), soil amendments resulting in microbial communities exhibiting significant differences in composition between the fumigated and unfumigated controls at 4 weeks post-amendment were selected for use in an artificial re-infestation experiment. This investigation was designed to simulate pathogen re-infestation of soil following fumigation and involved the introduction of the oomycete pathogen Pythium ultimum into pots containing rootstocks cultivated in the select soil amendments. Those amendments included FUM + BjSa (4.4 t ha−1), SMC, LC, and IF. As in the first experiment, controls included (1) fumigated soil, (2) unfumigated soil, and (3) unfumigated soil + BjSa (4.4 t ha−1). In order to simulate pathogen re-infestation following fumigation and test the ability of the apple rhizosphere microbiome to limit pathogen re-infestation, a mycelial fragment/spore suspension of isolate IR22 was prepared for use as a soil inoculum, as previously described [13,27,28]. Due to limited new root growth at 4 weeks, G.11 rootstocks were grown in 2.5 L pots containing the select soil amendments for 8 weeks prior to inoculation. Rhizosphere soil was then sub-sampled for DNA isolation/microbial community composition analysis as described above. Bulk soil was also collected in order to assess postharvest pathogen abundance at this time. DNA was extracted from 10 g of soil from each pot using the DNeasy Power Max Soil Kit (Qiagen). ITS amplicon sequencing was then conducted as described above. As stated above, soil infestation was conducted 8 weeks post-rootstock planting. The spore/mycelial fragment solution, in a volume of 300 mL per pot, was applied to soil planted with G.11 apple rootstocks to attain a final concentration of 300 propagules g−1 soil. In general, Pythium spp. populations range from 60 to 500 propagules in the orchard systems of Central Washington [13,29]. Bulk soil samples were collected immediately after inoculation to confirm the soil-established density of IR22. The experiment was harvested 1 month after inoculation.

2.11. Quantification of P. ultimum in Soil

In order to verify that P. ultimum IR22 levels were similar in all pots, DNA was extracted from bulk soil collected 24 h post-inoculation. P. ultimum quantification was conducted using a previously established species-specific qPCR assay [29]. Pathogen density in rhizosphere soil was also quantified upon rootstock harvest using the same assay. Kruskal–Wallis followed by Dunn’s multiple comparisons test was conducted between the fumigation alone control soil and all other soil treatments.

3. Results

3.1. Amendment Impacts on Soil Health Properties

In order to explore amendment-based changes in the abiotic health of fumigated orchard soil, a variety of metrics (Table 3) were used as the input for a principal components analysis (calculated on the correlation matrix of the dataset). The data from the fumigated alone control soil (collected at harvest) and that of all seven fumigated and amended treatments were then “projected” onto a two-dimensional scatter plot (Figure 1). PC1 and PC2 accounted for approximately 87% of the total variation. The more separation among treatments along PC1 (~75% of the variation), the more different they are. Relative to the fumigated control, fumigated soil amended with CCM or LC led to the greatest overall change in abiotic soil health characteristics; they both appear on the right side of PC1, while all other samples (including the fumigated control) appear on the left. The green lines indicate which factors influenced the variation and show correlations between the factors. For example, nitrate and EC are so tightly correlated along PC1 that their individual vector lines are hard to distinguish. This suggests that nitrates made up most of the soluble salts in the EC readings. Although CCM, LC, and SMC soil amendments resulted in the largest increases in soil organic matter (by 0.5–1.2%), both FUM + CCM and FUM + LC had high EC readings of 3.29 and 1.56 mmhos/cm, respectively. Most notably, the FUM + CCM treatment resulted in an EC value much higher than that predicted and was above the damage threshold for apple/pear (1.7 mmho/cm) [14].
The high level of nitrate (277 mg/kg) in the FUM + CCM treatment clearly had a negative impact on plant growth as none of the trees survived. Also, CCM and LC soil amendments resulted in the highest (7.8) and lowest (5.2) pH values, respectively (Table 3). Compost analysis indicated that CCM was moderately alkaline (pH = 8.5), typical of manure-based composts (Table S1). In general, high pH compost should be avoided on soils which are already above the optimum pH for tree fruit (optimum pH = 6–6.5) [14]. In comparison, the data in Figure 1 indicated that BjSa SM (2.2 t), B. napus SM (4.4 t), SMC, and IF soil amendments altered the chemical and physical properties of fumigated soil in similar ways, forming a relatively compact cluster of points in the upper left quadrant of the plot. Relative to the fumigated control, these samples evidenced increased water holding capacity, pH levels, and C:N ratios. In all treatments, however, C:N ratios were ≤10.

3.2. ARD Bioassay

Unfumigated SRO (plot 12b) and fumigated replant soils (plot 14b) were used in a bioassay to ensure disease control was in fact obtained in fumigated soil. The total plant biomass (Figure S1) was significantly higher in fumigated and pasteurized replant soil than in unfumigated (i.e., replant) soil, providing strong evidence of disease control in the fumigated soil. Upon harvest, the average number of P. penetrans recovered per gram of root tissue was 183 for plants cultivated in replant soil. By comparison, not a single P. penetrans was identified in the roots of apple seedlings cultivated in fumigated or pasteurized replant soil.

3.3. Amendment-Based Changes to Rhizosphere Microbial Community Composition (Four Weeks Post-Planting)

Bacterial community composition in the rhizosphere was dissimilar among all treatment groups (1-Way ANOSIM; p ≤ 0.05). However, PCoA plots indicated that insect frass and BjSa SM (2.2 t ha−1) were relatively less successful in their ability to significantly alter the bacterial composition of the fumigated rhizosphere microbiome and “steer” the community in a positive direction four weeks post-planting (Figure 2). Fumigated soil amended with the BjSa SM (4.4 t ha−1) treatment (in dark blue) moved the bacterial community closest to that of the disease-suppressive control treatment (pink). Shiitake mushroom compost and liquid chitin soil amendments (in teal and grey) also worked well at moving the bacterial community away from known disease-conducive states but in a different direction. Although statistically dissimilar, fumigated and unfumigated replant control soils were most similar in terms of community composition at this time point.
ANOSIM among fungal communities indicated that all treatments significantly altered rhizobiome composition relative to the fumigated and unfumigated replant control treatments. Fungal community composition in the rhizosphere did not differ significantly between the target control treatment and FUM + LC, between FUM + LC and FUM + SMC, or between FUM + B. napus SM (4.4 t ha −1) and FUM + BjSa SM (4.4 t ha−1). When examined using PCoA, the IF amendment (yellow) was highly similar to the “target” treatment while FUM + BjSa SM (2.2 t) (in light blue) was most similar to that of the fumigated alone control treatment (Figure 3).

3.4. In-Depth Assessment of Microbial Community Sequence Data

Fungi: In all amended treatments, potentially beneficial fungi with activity against specific ARD pathogens were significantly enriched relative to the fumigated control (Table S2). In the target treatment, Humicola, Emericellopsis, and Gelasinospora brevispora represented approximately 50% of the total fungi. Chaetomium spp., however, became dominant in the frass treatment (60% of the total fungi; Table S2). Chaetomium sp. made up only 12% of the fungi in the rhizosphere soil from the fumigated control alone. All of these fungal groups include members possessing the potential to control replant pathogens (including P. ultimum and R. solani) and are likely to be beneficial in terms of disease control.
In FUM+ BjSa (4.4 t ha−1 and 2.2 t ha−1), FUM + SMC, and FUM + LC, Fusarium oxysporum became significantly enriched relative to the fumigated control and represented over 10% of the relative abundance in these treatments. Fusarium oxysporum is commonly associated with apple roots in orchards and is considered to be non-pathogenic toward apples [30] All three SM treatments led to significant increases in the relative abundance of Mortierella spp., a group of fungi generally well-known for their ability to provide multiple beneficial functions to a variety of plants, including the production of plant growth-promoting compounds (Table S2). The only treatments in which Hypocrea (i.e., Trichoderma) was enriched relative to the fumigated control were FUM + SMC and FUM + LC. These genera have commonly been reported to increase in response to the seed meal amendments [1] and contain multiple members known to be antagonistic toward both ARD fungal and oomycete pathogens. That said, the majority of fungi (53%) in FUM + LC rhizospheres were most similar to Sordaria tomento-alba, a species that is not well described in the literature but is not reported as a pathogen of apples.
Finally, sequence data indicated that the relative abundance of fungal replant pathogens in the rhizosphere post-fumigation was low (<1%). However, relative percentages of Ilyonectria robusta were significantly lower than those of fumigated control soil in all treatments (with the exception of FUM + IF) (p < 0.05, Table S3). The relative percentage of Cylindrocarpon sp. was also significantly reduced in FUM + IF, FUM + SMC, and Replant + BjSa (4.4 t ha−1) control treatments relative to those of fumigated control soil by ~10x Together, these results suggest that all soil amendments had at least some ability to reduce fungal pathogen activity 1 month post-planting.
Bacteria: With respect to changes in the bacterial community, all soil amendments led to an increase in the relative abundance of Bacillales, a group which dominated the “target” treatment (>50%) (Figure 4). A variety of Bacillus species possess cellulolytic capabilities (i.e., the potential for oomycete control) [31,32,33]. In addition, a number of species exhibit biocontrol activity against specific apple replant pathogens [34,35,36] as well as postharvest pathogens of apples [37,38,39,40]. Members of this group may also positively influence apple plant growth in other ways [41,42]. Bacillales were 40% of the total bacteria in the rhizosphere of FUM + BjSa SM (4.4 t ha−1), which moved the community closest to that of the “target” treatment. BjSa SM (2.2 t ha−1), by comparison, was much less effective at moving the community away from fumigation-alone or unfumigated replant control treatments (i.e., chronic disease states).
Other treatments that worked well at driving the bacterial community out of the post-fumigation state, but in a different direction than the BjSa SM (4.4 t ha−1) treatments, included FUM + SMC and FUM + LC. Shiitake mushroom compost (but not the liquid chitin product) appeared to have a proliferative effect on chitinolytic bacteria in the order Chitinophagales (family Chitinophagaceae) (Figure 4). The fact that the LC amendment did not result in a significant increase in chitinolytic bacteria was unexpected as the product would be expected to contain high levels of chitin. Instead, the treatment appeared to enrich a wide variety of bacteria within the orders Xanthomonadales (e.g., Dyella, Frateuria, Rhodanobacter spp.) and Burkholderiales, including B. xenovarans (Figure 4). Thus, LC appears to have increased the potential of the rhizobiome to utilize unique and complex compounds for growth (esp. environmental pollutants). However, as described above, the assessment of abiotic soil health characteristics indicated that LC is likely to be detrimental to both plant and soil health when used as a post-fumigation soil amendment.
Relative to fumigated controls, bacteria involved in nitrogen-cycling were also enriched in the FUM + SMC treatment. Interestingly, this soil amendment tended to support the growth of bacteria with the ability to fix nitrogen from the atmosphere, namely those within the order Hyphomicrobiales, family Rhizobiaceae (Figure 4). The SMC soil amendment was the only treatment which resulted in reduced levels of plant available NO3 in bulk soil (Table 3). Therefore, this group may have facilitated plant access to soil nitrogen. Taken together, this result suggests that the growth of the trees cultivated in FUM + SMC may have become nitrogen limited. SMC would be undesirable if N fertilization was the goal.

3.5. Bacterial and Fungal Rhizosphere Community Trajectory at Harvest (8 Weeks Post-Planting)

Many of the bacterial communities that had initially diverged away from disease-conducive states appeared to be shifting back by 2 months post-amendment (Figure 2 vs. Figure S2A). This may have partly been due to a depletion of nutritional resources in the soil over time. In comparison, bacterial communities in fumigated and replant rhizosphere soil stayed within a narrow region of community space regardless of timepoint, suggestive of “stable state” dynamics [25]. The trajectory of the bacterial rhizobiome associated with the LC amendment also appeared less variable over time, suggestive of an alternative stable state. In terms of fungal community dynamics, community configurations generally appeared to be less variable over time relative to the fumigated soil (Figure 3 vs. Figure S2B).

3.6. Impacts on Plant Fitness

The amount of wood produced during the growing period (trunk diameter) is an indicator of overall tree health. In general, with the exception of the replant-alone control (Figure 5; red), all amendments resulted in an increase (albeit non-significant) in mean trunk diameter relative to the fumigation alone control soil (black). Both the nutritional and microbially-mediated aspects associated with these treatments are likely to bolster plant performance to values higher than those that would be expected with fumigation alone. For example, mustard seed meal possesses approximately 6% N, 2% S, 2% P, and 1% K. Notably, insect frass was the only treatment that resulted in a significant increase in trunk diameter (from planting to harvest) relative to the fumigated control (Kruskal–Wallis Test followed by Dunn’s Multiple Comparisons Test; p = 0.03). This result suggests that insect frass may benefit the growth of young trees in fumigated soil. No significant differences were identified in leader shoot length between the unamended fumigated soil control treatment and all other soil treatments (Kruskal–Wallis Test followed by Dunn’s Multiple Comparisons Test, p = 0.64). Although not significant, it should also be noted that plants cultivated in the SMC treatment allocated more biomass to root tissue relative to shoot tissue than any other treatment (Figure S4). A more robust root system may be beneficial toward facilitating increased tolerance to drought and/or replant pathogens.

3.7. (Artificial) Re-Infestation Experiment

In order to determine the ability of the altered microbiome to inhibit pathogen re-infestation of fumigated orchard soil, the most promising soil amendments (BjSa (4.4 t ha−1), SMC, LC, and IF) were selected for use in a subsequent experiment. These four soil amendments led to compositional changes in bacterial and fungal community space similar to those in the initial microbial “steering” investigation although the fungal community shift in FUM + IF was not as dramatic (Figure S3). In the microbial steering experiment, a single out (Chaetomium sp.) became dominant in the frass treatment (60% of the total fungi). In this experiment, two members of the Chaetomiaceae (Chaetomium globosum; 19.8% and Corynascus verrucosus; 4%) were significantly enriched in FUM + IF relative to fumigated controls prior to pathogen infestation.
No significant differences were identified between any of the treatments in terms of the initial inoculum densities of P. ultimum in bulk soil collected 24 hrs post-inoculation (5–7 pots per treatment) (one-way ANOVA followed by Tukey’s Multiple Comparisons Test; data natural-logtransformed). Upon harvest (1 month after inoculation), however, P. ultimum pathogen density in rhizosphere soil was significantly elevated in fumigated soil amended with insect frass relative to the fumigated control (Kruskal–Wallis Test followed by Dunn’s Multiple Comparisons Test; p = 0.003) (Figure 6). This result suggests that insect frass may serve as a nutritional/carbon source for Pythium spp. No other significant differences were identified. No treatment resulted in a statistically significant increase in trunk diameter (from planting to harvest) or leader shoot length relative to the fumigated control. However, a positive trend was observed for FUM + BjSa (4.4 t ha−1) as the treatment resulted in the greatest increase in trunk diameter and leader shoot length.

3.8. Assessment of Postharvest Pathogens in Bulk Soil

In the second experiment, bulk soil was collected prior to inoculation with P. ultimum. DNA was then extracted and the relative abundance of several postharvest pathogens including Penicillium spp. and Alternaria spp., among others, were assessed via sequence-based analysis. Of the pathogens assessed, the majority are known to be problematic in the Pacific Northwest, especially Washington State (Achour Amiri pers comm). One of the most problematic postharvest pathogens in the region is Penicillium expansum, which causes blue mold and produces the mycotoxin patulin [43]. Results suggest that this organism may have been stimulated by insect frass (Table 4). Overall, the postharvest pathogenic fungi identified in the bulk soil from this experiment (7/22 species) comprised a small percentage of the fungal community (Table 4). Potential postharvest fungal pathogens not identified in bulk soil are listed in Table S4 (e.g., Botrytis cinerea and Mucor piriformis).
A handful of postharvest pathogens of interest were detected and identified as being significantly different in fumigated control vs. amended soil (Table 4). For example, the relative abundance of Alternaria alternata (a fungus which is commonly found in orchard soil and is a causative agent of Alternaria rot) was significantly reduced relative to unfumigated orchard soil in all treatments except FUM + IF. The only treatment resulting in a significant decrease in A. alternata relative to the fumigated control was the LC amendment. In almost all treatments, Cladosporium cladosporioides was also present in low abundance and this fungus was not detected in bulk soil amended with shiitake mushroom compost. Finally, it is worth noting that Aspergillus niger, a causative agent of postharvest black mold in apple, was elevated in replant soil amended with 4.4 t ha−1 BjSa SM, albeit not significantly. Regardless, A. niger is not a significant postharvest pathogen in Washington State (A. Amiri pers comm).

4. Discussion

Trees in modern orchard systems are likely to be more vulnerable to multiple stress factors, including disease and climatic stress, than trees in traditional systems that did not utilize dwarfing rootstocks. In many ways, high-density plantings (1000–6000 trees ha−1) are at greater risk to the effects of pests/pathogens than previously low-density orchard systems (70–100 trees ha−1). Thus, integrated control measures for reducing the infection frequency of pests, both above and below-ground, are becoming increasingly important. ARD can cost growers up to 70,000 USD per acre in lost returns in the absence of effective treatment [44]. An amendment-mediated control of soilborne pathogens in fumigated replant soil may improve the ability of soil to defend against reinfestation of fumigated soil by root infecting and potential postharvest fungal pathogens that reside in soil.
In this study, we evaluated the potential of various organic soil amendments to “steer” resident rhizosphere microbiome community configurations away from known disease-conducive states following fumigation and to improve abiotic soil health characteristics. When incorporated into fumigated soil, composted chicken manure and liquid chitin led to high salinity, as measured by electrical conductivity. This metric was tightly correlated with soil nitrate levels. Most notably, excessive nitrate resulting from CCM (277 mg/kg) resulted in the death of all trees. Therefore, the use of composted chicken manure as a post-fumigation soil amendment (even when incorporated at a relatively low rate 6 weeks prior to planting) is not recommended. Amendment-induced salt toxicity in newly established orchards is generally not an issue when composted (or raw) chicken manure is used as a top dressing. However, nutrients are less likely to be root-absorbed when CCM is applied in this manner. This is especially true for NO3-N, which is easily leached from soil by rain or over-irrigation. Nitrate accumulation in surface or groundwater poses significant long-term risks to the environment and human health. Therefore, the use of CCM as a top dressing is also unadvisable.
Findings from the current study suggest that Brassica seed meals, shiitake mushroom compost (SMC), and insect frass (IF), although more expensive than CCM and LC, are likely to benefit multiple soil health characteristics when used as a post-fumigation soil amendment. These materials altered the chemical and physical properties of fumigated replant soil in similar ways including an increased pH, C:N ratio, and water holding capacity.
It was previously found that in orchard field trials with replant disease, SM amendment rates of 4.4 and 6.6 t ha−1 significantly reduced fungal and bacterial diversity indices relative to the no-treatment control [1,2]. This is important because diminished diversity together with selective augmentation of specific components of the rhizosphere microbiome have consistently been associated with a corresponding disease control. In the study by Wang and Mazzola (2019) [2], BjSa SM application at 2.2 t ha−1 was not as effective or predictable as 4.4 t ha−1 or soil fumigation. At 2.2 t ha−1, the M.26 trunk diameter was no different from that of the untreated control whereas G.41 did not attain ‘fumigation level’ fruit yields [2]. Although all three application rates have been shown to amplify several potentially plant-beneficial bacteria and fungi in the apple rhizosphere one year post-planting, the overall composition of the rhizosphere microbiome of M.26 or G.41 was less affected by the 2.2 than by the 4.4 t ha−1 rate [2].
In this study, the use of a BjSa SM at an application rate of 2.2 t ha−1 was examined for its potential to be a more cost effective approach when used as a post-fumigation management strategy. Our results suggest that 2.2 t ha−1 is not as effective as 4.4 t ha−1 at promoting changes in the soil microbial community following fumigation. Although fumigation is used to suppress soilborne pathogens, it affects the entire resident community, including beneficial microbes. A recent study utilized network analysis to illustrate how soil fumigation decreased interactions within the soil microbial community and created empty niches [45]. Surviving resident populations exist in a disabled microbial network (at least temporarily) and may not be as responsive to SM-based remodeling as unfumigated soil. Therefore, use of BjSa SM as an alternative to (rather than in addition to) fumigation is more effective from both cost and disease management standpoints.
In all soil amendment treatments, potentially beneficial fungi with activity against specific ARD pathogens were significantly enriched in the rhizosphere soil relative to the fumigated control. The IF amendment, however, not only moved the fungal community close to that of the “target” treatment but also performed best in terms of tree growth. Based on these data, insect frass appears to be an organic amendment source with a high potential for positive outcomes in the field. An important caveat is that IF failed to effectively re-shape the bacterial community and provide protection against P. ultimum. Taken together, these results serve as a reminder that microbiome-mediated plant protection is governed by a microbial consortium rather than individual elements of the soil microbial community.
ARD is a particularly complex disease phenomenon as it is caused by a variety of fungal, oomycete, and nematode root pathogens acting synergistically (rather than individually) [13]. Different pathogens may dominate (i.e., contribute more to ARD) depending on the location or season and individually these pathogens are likely to be affected differently by different amendments. For example, studies show B. napus SM is stimulatory toward Pythium spp. in orchard soil and apple roots but suppressive to root infection by Rhizoctonia spp. and Pratylenchus penetrans [26,46]. In another study, mushroom compost soil amendment significantly reduced damping off in pine seedlings by Rhizoctonia solani AG2 [47], a fungus closely related to the apple root pathogen Rhizoctonia solani AG-5. The current study examined only a single pathogen, P. ultimum. Thus, it is important to bear in mind that certain soil amendments selectively amplify or suppress populations of specific pathogens.
In this study, the ability of select amendments to restrict Pythium re-infestation following fumigation was examined. It was expected that fumigated soil + select soil amendments would be more resilient to re-infestation than fumigated soil alone. No treatment significantly improved the ability of the rhizosphere to suppress P. ultimum post-fumigation. The FUM + BjSa (4.4 t ha−1) treatment, however, showed the most promise as the pathogen was only detected in two out of six plants (Figure 6). Additionally, P. ultimum was unable to effectively colonize plants cultivated in the replant + BjSa (4.4 t ha−1) control treatment. This amendment has been shown to suppress root densities of Pythium spp. in a manner equivalent to fumigation while also supporting a rhizosphere microbiome associated with multi-year ARD suppression [2]. As mentioned previously, insect frass resulted in P. ultimum levels being significantly elevated relative to the fumigated control. P. ultimum infestation of fumigated (but not unfumigated soil) may be explained by an inability of the introduced pathogen to compete with indigenous microorganisms (including other replant pathogens) which became established in the root and rhizosphere prior to inoculation. Similarly, multiple soilborne pathogens (including Pythium species) rapidly reinfest fumigated soil and are commonly found in populations higher than that observed prior to treatment in the field [1,2]. Attempts were also made to quantify P. ultimum levels in root tissue. Results were highly variable, however. Intact tissues infected with this pathogen (e.g., root hairs) are generally ephemeral and are commonly lost through the harvesting process. In addition, root samples contain inhibitory substances (e.g., phenolics and humic acids) which can be co-extracted with nucleic acids and lead to variability in qPCR. In terms of plant growth, we were unable to differentiate treatment growth responses and suspect that the experimental duration was not long enough to achieve sufficient root development.
The ability of select amendments to reduce potential postharvest pathogens in bulk soil following fumigation was also examined as part of the re-infestation experiment. The fungi Chaetomium globosum and Hypocrea virens (i.e., Trichoderma) have both been shown to exhibit biocontrol activity against A. alternata [48]. C. globosum, represented roughly 4% (bulk soil) and 20% (rhizosphere soil) of the total fungal community identified in the FUM + IF treatment. The increase in relative abundance of A. alternata in FUM + IF (relative to fumigated and unfumigated controls) was therefore unexpected. It may be that C. globosum levels in bulk soil were not high enough to effectively inhibit A. alternata. It is also notable that the only treatment resulting in a significant decrease in A. alternata relative to the fumigated control was the LC amendment. This may be explained by the elevated abundance of H. virens which represented 62% of the total fungal community in the bulk soil from the LC amendment but only 2% in the fumigated alone control.
Although stimulatory toward Pythium, the results of these experiments suggest that insect frass is a promising treatment for boosting the growth of young apple trees and improving multiple aspects of soil health post-fumigation. Although some hurdles remain to be addressed, we think it is worth continuing to investigate this material. Logical next steps include a more comprehensive evaluation as to how the amendment affects additional components of the ARD disease complex, optimization of amendment rates, and/or determination of which rootstocks and soil types respond best to the amendment. It is unclear as to how these advantages/disadvantages would be expressed in the field. Following future studies such as these, field experiments could be conducted to determine whether apple yields could be increased in fumigation + amendment relative to fumigation alone over an extended period of time.

5. Conclusions

Many host-associated communities (e.g., the human gut microbiome) can act as potential controllers of wellness and disease [49]. Microbial communities are not static over time, however, and this makes the engineering of host-associated microbiomes especially challenging. Results from this study ground support for the use of soil amendments as an intervention strategy for “steering” the soil and rhizosphere microbiome in more beneficial and/or prophylactic directions following fumigation. Many of the soil amendments used here evidenced compositional changes that encouraged desired outcomes (i.e., an increase in taxa with the potential to suppress disease progression). Going forward, the lens of ARD research must take in the complex network of its nature. This means focusing in on direct assessments of functional changes in the microbiome in response to management practices (i.e., disease control and plant nutrient uptake) as well as developing a more systematic understanding of the amendment components which functionally promote an effective microbiome. All are essential for the rehabilitation of a functional soil microbiome and the establishment of a resilient rhizobiome.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agriculture13112104/s1. Figure S1. Effect of soil treatment on growth of 4 week old Gala apple seedlings as measured by total plant biomass; Figure S2. Principal coordinates analysis of bacterial (A) and fungal (B) community composition in G.11 apple rhizospheres 8 weeks post-planting at the order level; Figure S3. Principal coordinates analysis of bacterial (A) and fungal (B) community composition in select treatments 8 weeks post-planting (prior to P.ultimum inoculation) at the order level.; Figure S4. Simple linear regression of plant root biomass to shoot biomass at harvest (8 weeks post planting). Table S1. Results of analyses conducted for evaluation of composted chicken manure (CCM) and shitake mushroom compost (SMC) prior to use as soil amendments; Table S2. Potentially beneficial fungi significantly enriched in rhizospheres of G.11 apple rootstocks cultivated in treated soils relative to fumigated alone control soil [50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67]; Table S3. Median relative percentages of fungal replant pathogens identified by the Two-Part test as being significantly different in treated soil relative to those of fumigated control soil; Table S4. List of potential post-harvest fungal pathogens of apple not detected in bulk soil.

Author Contributions

Conceptualization, Methodology, Funding Acquisition, Supervision, Data Analysis, Writing: T.S.; Supervision, Writing: M.M.; Data analysis, Writing: C.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded, in part, through a grant to T. Somera from the Washington State Tree Fruit Research Commission; Agreement No. 58-2094-1-003.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The datasets generated and analyzed during the current study are available in the Figshare repository (10.6084/m9.figshare.24476923) and include 16S rRNA and ITS OTU read count tables from the Microbial Steering Experiment and from the (Artifical) Re-Infestation Experiment. Any other data generated or analyzed during this study are included in this published article and its Supplementary Information File or are available from the corresponding author upon reasonable request.

Acknowledgments

We thank David Granatstein for helpful advice and suggestions during proposal development. We also thank Achour Amiri for postharvest pathogen assessment guidance.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Principal component analysis of abiotic soil health characteristics in fumigated orchard soil (Fum control), fumigated soil amended with BjSa seed meal (2.2 and 4.4 t ha−1), shiitake mushroom compost (SMC), composted chicken manure (CCM), liquid chitin (LC), insect frass (IF), or B. napus seed meal (4.4 t ha−1) at 3 months post-planting. All soil characteristics listed in Table 3 (with the exception of the individual cations influencing cation exchange capacity) were used in the analysis (Past v.3).
Figure 1. Principal component analysis of abiotic soil health characteristics in fumigated orchard soil (Fum control), fumigated soil amended with BjSa seed meal (2.2 and 4.4 t ha−1), shiitake mushroom compost (SMC), composted chicken manure (CCM), liquid chitin (LC), insect frass (IF), or B. napus seed meal (4.4 t ha−1) at 3 months post-planting. All soil characteristics listed in Table 3 (with the exception of the individual cations influencing cation exchange capacity) were used in the analysis (Past v.3).
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Figure 2. Principal coordinate analysis of bacterial community composition in G.11 apple rhizospheres 4 weeks post-planting at the order level. Each point represents bacterial community sequence data generated from an individual soil sample. Filled shapes represent the convex hull of replicate samples associated with plants cultivated in the different treatments as labeled. The ‘bullseye’ symbol indicates the target (i.e., disease-suppressive) control treatment or one direction to move toward. Microbial community configurations which are disease conducive (i.e., directions to move away from) are represented by the ‘do not’ symbol. None of the rootstocks planted into the fumigated orchard soil amended with chicken manure compost (CCM) had any signs of new root or shoot growth. Therefore, CCM is not included in the figure. The term “replant” refers to unfumigated soil.
Figure 2. Principal coordinate analysis of bacterial community composition in G.11 apple rhizospheres 4 weeks post-planting at the order level. Each point represents bacterial community sequence data generated from an individual soil sample. Filled shapes represent the convex hull of replicate samples associated with plants cultivated in the different treatments as labeled. The ‘bullseye’ symbol indicates the target (i.e., disease-suppressive) control treatment or one direction to move toward. Microbial community configurations which are disease conducive (i.e., directions to move away from) are represented by the ‘do not’ symbol. None of the rootstocks planted into the fumigated orchard soil amended with chicken manure compost (CCM) had any signs of new root or shoot growth. Therefore, CCM is not included in the figure. The term “replant” refers to unfumigated soil.
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Figure 3. Principal coordinate analysis of fungal community composition in G.11 apple rhizospheres 4 weeks post-planting at the order level. Each point represents fungal community composition. Filled shapes represent the convex hull of replicate samples associated with plants cultivated in the different treatments as labeled. The ‘bullseye’ symbol indicates the target (i.e., disease-suppressive) control treatment or one direction to move toward. Microbial community configurations that are disease conducive (i.e., directions to move away from) are represented by the ‘do not’ symbol. None of the rootstocks planted into the fumigated soil amended with chicken manure compost (CCM) had any signs of new root or shoot growth. Therefore, CCM is not included in the figure. The term “replant” refers to unfumigated soil.
Figure 3. Principal coordinate analysis of fungal community composition in G.11 apple rhizospheres 4 weeks post-planting at the order level. Each point represents fungal community composition. Filled shapes represent the convex hull of replicate samples associated with plants cultivated in the different treatments as labeled. The ‘bullseye’ symbol indicates the target (i.e., disease-suppressive) control treatment or one direction to move toward. Microbial community configurations that are disease conducive (i.e., directions to move away from) are represented by the ‘do not’ symbol. None of the rootstocks planted into the fumigated soil amended with chicken manure compost (CCM) had any signs of new root or shoot growth. Therefore, CCM is not included in the figure. The term “replant” refers to unfumigated soil.
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Figure 4. Stacked bar graph showing relative percentages of select bacterial taxa at the order level which were identified by the two-part test as being significantly different (p < 0.05) relative to fumigated control rhizosphere soil at 4 weeks post planting. FUM = fumigated alone control, SMC = shiitake mushroom compost, LC = liquid chitin, IF = insect frass. None of the rootstocks planted into the fumigated soil amended with chicken manure compost (CCM) had any signs of new root or shoot growth. Therefore, CCM is not included in the figure. The term “replant” refers to unfumigated soil.
Figure 4. Stacked bar graph showing relative percentages of select bacterial taxa at the order level which were identified by the two-part test as being significantly different (p < 0.05) relative to fumigated control rhizosphere soil at 4 weeks post planting. FUM = fumigated alone control, SMC = shiitake mushroom compost, LC = liquid chitin, IF = insect frass. None of the rootstocks planted into the fumigated soil amended with chicken manure compost (CCM) had any signs of new root or shoot growth. Therefore, CCM is not included in the figure. The term “replant” refers to unfumigated soil.
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Figure 5. Increase in trunk diameter from planting to harvest. Different lowercase letters indicate significantly different means and represent statistical comparisons between the fumigated alone control soil (FUM Alone; black bar) and all other soil treatments (Kruskal–Wallis Test followed by Dunn’s Multiple Comparisons Test; p < 0.05). The absence of a lower-case letter indicates no significant difference from the fumigated alone control. SMC = shiitake mushroom compost, IF = insect frass, LC = liquid chitin, BjSa = B. juncea/S. alba seed meal. None of the rootstocks planted into the fumigated soil amended with chicken manure compost (CCM) had any signs of new root or shoot growth. Therefore, CCM is not included in the figure. Bars represent the standard error of the mean. Replant alone (i.e., unfumigated) control; red bar.
Figure 5. Increase in trunk diameter from planting to harvest. Different lowercase letters indicate significantly different means and represent statistical comparisons between the fumigated alone control soil (FUM Alone; black bar) and all other soil treatments (Kruskal–Wallis Test followed by Dunn’s Multiple Comparisons Test; p < 0.05). The absence of a lower-case letter indicates no significant difference from the fumigated alone control. SMC = shiitake mushroom compost, IF = insect frass, LC = liquid chitin, BjSa = B. juncea/S. alba seed meal. None of the rootstocks planted into the fumigated soil amended with chicken manure compost (CCM) had any signs of new root or shoot growth. Therefore, CCM is not included in the figure. Bars represent the standard error of the mean. Replant alone (i.e., unfumigated) control; red bar.
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Figure 6. Pythium ultimum density in rhizosphere soil upon rootstock harvest (1 month after inoculation). Different lowercase letters indicate significantly different means and represent statistical comparisons between the fumigation alone control soil (FUM Alone) and all other soil treatments (Kruskal–Wallis Test followed by Dunn’s Multiple Comparisons Test; p < 0.05). The absence of a lower-case letter indicates no significant difference from the fumigated alone control. Bars represent the standard error of the mean; SMC, shiitake mushroom compost; LC, liquid chitin; IF, insect frass. Replant Alone refers to the unfumigated control soil.
Figure 6. Pythium ultimum density in rhizosphere soil upon rootstock harvest (1 month after inoculation). Different lowercase letters indicate significantly different means and represent statistical comparisons between the fumigation alone control soil (FUM Alone) and all other soil treatments (Kruskal–Wallis Test followed by Dunn’s Multiple Comparisons Test; p < 0.05). The absence of a lower-case letter indicates no significant difference from the fumigated alone control. Bars represent the standard error of the mean; SMC, shiitake mushroom compost; LC, liquid chitin; IF, insect frass. Replant Alone refers to the unfumigated control soil.
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Table 1. Estimated costs for soil amendments. Costs were calculated according to the rates evaluated in this study and are based on the following assumptions: (1) amendments are tilled to a depth of 20 cm and (2) tree rows occupy approximately one-third of the total orchard. For the composted chicken manure, the cost was based on an application rate appropriate for new trees. Estimates are for materials only and do not include labor/application/delivery costs unless otherwise stated. Considering that any orchard management program is a balance of costs and benefits, approximate expenses for weed control and fertilizer are also included.
Table 1. Estimated costs for soil amendments. Costs were calculated according to the rates evaluated in this study and are based on the following assumptions: (1) amendments are tilled to a depth of 20 cm and (2) tree rows occupy approximately one-third of the total orchard. For the composted chicken manure, the cost was based on an application rate appropriate for new trees. Estimates are for materials only and do not include labor/application/delivery costs unless otherwise stated. Considering that any orchard management program is a balance of costs and benefits, approximate expenses for weed control and fertilizer are also included.
Management StrategyAnnual Cost (USD)
Conventional soil fumigation700–1700
Weed control (labor + materials; 2 applications per year)200–1012
Fertilizer (labor + materials)60α
ab Brassica seed meal (4.4 t ha−1)1380–3482
ab Brassica seed meal (2.2 t ha−1)700–1700
a Shiitake mushroom compost (2% v:v) *940
Insect (mealworm) frass (3.2 t ha−1)920
a Composted chicken manure (1.2 t ha−1)50
a Chitin-based amendment (20–40 L ha−1)20–40
a Need for chemical fertilizers reduced. b Need for weed control reduced. * based on dry weight.
Table 2. Application rates of organic amendments used in this study.
Table 2. Application rates of organic amendments used in this study.
TreatmentApplication RateNotes
Fumigated replant soil NADisease-conducive control
Unfumigated replant soil NADisease-conducive control
Unfumigated replant soil + BjSa SM 4.4 ton ha−1Disease-suppressive control
FUM + BjSa SM4.4 ton ha−1
FUM + BjSa SM2.2 ton ha−1
FUM + B. napus SM4.4 ton ha−1
FUM + SMC2% v/vRate estimated based on cost
FUM + IF3.2 ton ha−1Rate recommended by mfr.
FUM + CCM1.7 ton ha−1Rate estimated based on EC
FUM + LC20 L ha−1Rate recommended by mfr.
Note: For Brassica seed meal (SM) applications, 2.2 and 4.4 tons per hectare equates to 1 and 2 tons per acre, respectively; for insect frass (IF), 3.2 tons per hectare equates to 1.5 cups per ft−3 soil; for Shiitake mushroom compost (SMC) application, 2% v:v is based on the dry weight of the material; FUM refers to fumigated replant soil; LC, liquid chitin; CCM, composted chicken manure; EC, electrical conductivity; NA, not applicable; mfr., manufacturer.
Table 3. The effect of the different soil amendments on the chemical and physical properties of fumigated orchard soil. These metrics are for bulk soil collected 3 months post-planting. All analyses were conducted by Soiltest Farm Consultants (Moses Lake, WA, USA). FUM refers to fumigated replant soil; WHC = water holding capacity; OM = organic matter.
Table 3. The effect of the different soil amendments on the chemical and physical properties of fumigated orchard soil. These metrics are for bulk soil collected 3 months post-planting. All analyses were conducted by Soiltest Farm Consultants (Moses Lake, WA, USA). FUM refers to fumigated replant soil; WHC = water holding capacity; OM = organic matter.
TreatmentpHEC §CEC †Na #Ca #Mg #K #WHC (in/ft)OM (%)Total N (%)Total C (%)C:N NO3 ‡NH4 ‡SO42 ‡ P ‡K ‡B ‡Zn ‡Mn ‡Cu ‡Fe ‡
Fumigated replant soil *7.200.298.801.0084.1022.5016.801.511.700.090.697.6014.503.6017245780.236.202.700.9017
Fumigated replant soil6.000.309.101.3056.7016.2011.101.281.400.080.658.707.901.1028163930.176.001.700.6024
Unfumigated replant soil6.200.6811.201.1057.7016.1014.801.781.600.110.969.0023.101.6064416450.3010.501.702.6027
Unfumigated replant soil + BjSa SM (4.4 t ha−1)5.900.5910.801.2064.0018.1017.401.751.600.110.999.3044.902.8080417370.3510.304.503.0037
FUM + BjSa SM (2.2 t ha−1)7.000.458.002.7072.4021.8016.901.401.400.100.899.3034.8013.5038405310.247.102.201.1014
FUM + BjSa SM (4.4 t ha−1)6.600.747.804.9077.7025.8023.101.951.500.111.1210.2069.5014.3055407060.327.003.701.4017
FUM + B.napus SM (4.4 t ha−1) 6.600.538.201.5071.0021.4014.901.381.300.080.7910.1079.701.4032214770.256.602.600.7027
FUM + SMC 7.500.368.501.2071.5020.9014.101.411.900.090.899.702.504.5016234710.155.901.800.5013
FUM + IF 7.100.058.701.2066.1020.8014.401.501.600.080.789.3016.001.5023404890.176.901.200.6013
FUM + CCM 7.803.299.5029.5084.2039.5087.701.462.600.231.968.70277.00266.8027115732623.3525.1029.6015.0043
FUM + LC 5.201.569.302.7079.8020.9013.101.571.900.120.978.30147.508.50168444740.157.8021.501.0028
* Collected/analyzed prior to planting; § Electrical conductivity (mmhos/ cm); † Cation exchange capacity (meq/100 grams soil); # Percentage of cation exchange capacity (CEC); ‡ mg/kg.
Table 4. Relative abundance of potential postharvest fungal pathogens of apple detected in bulk soil amended with different treatments (8 weeks post-amendment).
Table 4. Relative abundance of potential postharvest fungal pathogens of apple detected in bulk soil amended with different treatments (8 weeks post-amendment).
Postharvest Fungal PathogenUnfumigatedUnfumigated + 4.4 t BjSa SM Fumigated FUM + IFFUM +SMCFUM+LCFUM + 4.4 t BjSa SM
Alternaria alternata0.17 *0.02 †0.05 †1.250.03 †0.01 *,†0.04 †
Aspergillus parasiticus000.010.010.01 †00
Aspergillus niger0.012.140.0100.010.010.01
Cladosporium cladosporioides0.040.060.020.020 *,†0.010.09
Glomerella cingulata0.01000000
Mucor circinelloides000000.070
Penicillium expansum0000.17†000
* Significantly different from the fumigated control (p < 0.05). † Significantly different from the unfumigated control (p < 0.05).
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Somera, T.; Mazzola, M.; Cook, C. Directing the Apple Rhizobiome toward Resiliency Post-Fumigation. Agriculture 2023, 13, 2104. https://doi.org/10.3390/agriculture13112104

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Somera T, Mazzola M, Cook C. Directing the Apple Rhizobiome toward Resiliency Post-Fumigation. Agriculture. 2023; 13(11):2104. https://doi.org/10.3390/agriculture13112104

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Somera, Tracey, Mark Mazzola, and Chris Cook. 2023. "Directing the Apple Rhizobiome toward Resiliency Post-Fumigation" Agriculture 13, no. 11: 2104. https://doi.org/10.3390/agriculture13112104

APA Style

Somera, T., Mazzola, M., & Cook, C. (2023). Directing the Apple Rhizobiome toward Resiliency Post-Fumigation. Agriculture, 13(11), 2104. https://doi.org/10.3390/agriculture13112104

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