Application sequence modulates microbiome composition, plant growth and apple replant disease control efficiency upon integration of anaerobic soil disinfestation and mustard seed meal amendment

Journal Pre-proof Application sequence modulates microbiome composition, plant growth and apple replant disease control efficiency upon integration of anaerobic soil disinfestation and mustard seed meal amendment Mark Mazzola, Danielle Graham, Likun Wang, Rachel Leisso, Shashika S. Hewavitharana PII:

S0261-2194(20)30058-2

DOI:

https://doi.org/10.1016/j.cropro.2020.105125

Reference:

JCRP 105125

To appear in:

Crop Protection

Received Date: 4 September 2019 Revised Date:

20 February 2020

Accepted Date: 23 February 2020

Please cite this article as: Mazzola, M., Graham, D., Wang, L., Leisso, R., Hewavitharana, S.S., Application sequence modulates microbiome composition, plant growth and apple replant disease control efficiency upon integration of anaerobic soil disinfestation and mustard seed meal amendment, Crop Protection (2020), doi: https://doi.org/10.1016/j.cropro.2020.105125. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

CRediT author statement Mark Mazzola: Conceptualization, Funding Acquisition, Supervision, Project Administration, Writing, Project Administration, Investigation, Visualization, Formal Analysis, Danielle Graham: Methodology; Investigation, Formal Analysis Likun Wang: Investigation Rachel Leisso: Methodology, Investigation Shashika S. Hewavitharana: Methodology, Investigation

1 1

Application sequence modulates microbiome composition, plant growth and apple replant

2

disease control efficiency upon integration of anaerobic soil disinfestation and mustard seed

3

meal amendment

4 5

Mark Mazzola1*, Danielle Graham1, Likun Wang2, Rachel Leisso1 and Shashika S. Hewavitharana3

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1

7

2

8

Wenatchee, Washington, USA

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3

USDA, Agricultural Research Service, 1104 N. Western Avenue, Wenatchee, Washington, USA Department of Plant Pathology, Washington State University, 1100 N. Western Avenue,

Horticulture and Crop Science Department, California Polytechnic State University, San Luis

10

Obispo, CA 93407

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*

12

Email address: [email protected]

Corresponding author:

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Declarations of interest: none.

2 15

Abstract

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Anaerobic soil disinfestation and mustard seed meal (MSM) amendments can provide effective

17

control of soil-borne diseases including apple replant disease. These measures rely on both

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chemical and biological modes of action to yield effective disease control and their integration

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may prove beneficial or, alternatively, deleterious to overall treatment efficiency when applied

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in concert. Potential outcomes of integrating ASD with MSM amendments were assessed by

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determining the effect of treatment application sequence and ASD carbon source on

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generation of allyl isothiocyanate (AITC) derived from Brassica juncea seed meal, structure of

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the rhizosphere and soil microbiome, control of apple replant pathogens, and plant growth. In

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bioassays conducted using ‘Gala’ apple seedlings, application of ASD or MSM treatments

25

independently was as effective or superior to all integrated treatments for the control of

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replant pathogens. Application of ASD prior to MSM amendment diminished the yield of AITC

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attained in response to soil incorporation of the Brassica juncea:Sinapis alba seed meal.

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Treatment application sequence had significant effect on structure of the bulk soil fungal and

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bacterial community. Correspondingly, treatment application sequence significantly altered

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plant growth performance when orchard grass was utilized as the ASD carbon input. At harvest,

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rhizosphere fungal but not bacterial community composition was significantly altered in treated

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soil relative to the control, and sequence of treatment application had significant effect on

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rhizosphere fungal community structure. Failure of integrated treatments to enhance overall

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replant disease control may have resulted from many factors including reduced generation of

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active metabolites, diminished activity of mechanisms functional in pathogen suppression, or

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the elevated accumulation and retention of phytotoxic chemistries, the latter which would

3 37

require extended plant back periods to circumvent. The findings indicate that under the

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experimental conditions employed, integration of ASD with MSM amendment is unlikely to

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yield additive or synergistic effects on apple replant disease control.

40 41 42

Keywords: Malus domestica Borkh., replant disease, anaerobic soil disinfestation (ASD),

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mustard seed meal (MSM), soil microbiome, terminal-restriction fragment length

44

polymorphism (T-RFLP)

4 45

1. Introduction

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High value fruit and vegetable specialty crops often rely upon pre-plant soil fumigation to

47

enable continuous crop cultivation while limiting exposure to potential damage resulting from

48

activity of soil-borne plant pathogens. Annual cropping systems such as strawberry are highly

49

reliant on such a disease control strategy, and system development was built upon the

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availability of effective fumigant chemistries (Wilhelm et al., 1974). While such generally was

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not a pre-determined tactic for use in perennial crops such as tree fruits, pre-plant soil

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fumigation has had an integral role in allowing the economically successful replanting of sites

53

that were previously cultivated to the same or closely related species (Mai and Abawi, 1981). In

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the absence of soil fumigation prior to establishing a new orchard on replant sites, trees

55

commonly exhibit poor growth and yield due to the activity of soil-borne plant pathogens, a

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phenomenon termed replant disease. In apple, replant disease has been documented

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throughout pome fruit production regions of the world and has been studied extensively

58

relative to its causation (Mazzola and Manici, 2012).

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Although consensus regarding causality of replant disease has not been achieved, a number

60

of studies have implicated a biological complex that includes fungi, oomycete and nematode

61

pathogens as the primary incitant of apple replant disease (Braun 1991; Jaffee et al., 1982a;

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1982b; Mazzola, 1998; Tewoldemedhin et al., 2011). Due to its composite nature, effective

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control of the causal pathogen complex inciting replant disease has consistently relied upon use

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of pre-plant soil fumigation. However, several emerging social and regulatory factors have

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motivated interest in the development of non-fumigant strategies for the control of apple

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replant disease. These factors include current needs of an expanding organic tree fruit

5 67

industry, implementation of limits in fumigant application methods or use, limited temporal

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benefit of the fumigation response and demonstration that alternative methods may have

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prolonged disease control activity (Mazzola et al., 2015; Wang and Mazzola, 2019a). The

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positive growth response resulting from pre-plant soil fumigation generally is limited to the

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initial year following orchard planting (Auvil et al., 2011; Robinson et al., 2014). The lack of a

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prolonged benefit of pre-plant soil fumigation to plant growth is likely due to the rapid

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recolonization of soils, and subsequent infestation of apple roots, by elements of the pathogen

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complex that incites replant disease including Pratylenchus penetrans and Pythium spp.

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(Mazzola et al., 2015; Wang and Mazzola, 2019a).

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Alternative methods examined for the control of replant disease are numerous and include,

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among others, utilization of disease tolerant apple rootstock genotypes (Kviklys et al., 2016),

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pre-plant soil incorporation of Brassicaceae seed meals (Mazzola and Brown, 2010; Mazzola et

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al., 2015), a variety of fertility and compost soil amendments (Slykhuis and Li, 1985; van Schoor

80

et al., 2009), anaerobic soil disinfestation (Hewavitharana and Mazzola, 2016a) and altered

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orchard planting structure (Rumberger et al., 2004). Host tolerance/resistance can be an

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economically effective and ecologically desirable strategy for the control of soil-borne diseases

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including apple replant disease. However, although apple rootstock genotypes possessing field

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level tolerance have been identified (Auvil et al., 2011; Robinson et al., 2012; Kviklys et al.,

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2016), these rootstocks are not immune from infection by causal agents of replant disease

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(Emmett et al., 2014; Wang and Mazzola, 2019a) and continue to exhibit significant growth and

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yield enhancement in response to pre-plant soil fumigation when established on replant

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orchard sites (Wang and Mazzola, 2019a). For instance, while the disease susceptible rootstock

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M.9 exhibited a 126% increase in trunk increment in response to 1,3-dichloropropene/

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chloropicrin soil fumigation over a two-year period, this same treatment resulted in a 79%

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increase in trunk increment for the highly tolerant G.935 rootstock over the same period at the

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same replant orchard site (Hewavitharana and Mazzola, 2016a). Similarly, in an additional field

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trial, 1,3-dichloropropene/chloropicrin soil fumigation resulted in significant increases in trunk

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diameter and yield for both Gala/M.26 (28.3% and 35.5%, respectively) and Gala/G.41 (28.6%

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and 64.2%, respectively) over two growing seasons (Wang and Mazzola, 2019a). Although

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resistance to Pythium ultimum has been reported for the rootstock G.935 in controlled

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environment experiments (Zhu et al., 2016), when examined under field conditions, P. ultimum

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root infestation was equivalent for the resistant (G.935) and a susceptible (M.9) rootstock

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genotype (Mazzola and Hewavitharana, 2019). Thus, the functional replant disease tolerance

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reported for the apple rootstocks G.935 and G.41 (Kviklys et al., 2016) is unlikely to provide

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optimal tree growth and yield performance in and of itself when used as a replant disease

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control tactic.

103

An array of amendment-based strategies has been evaluated as replacements to soil

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fumigants for promoting growth of apple on replant sites, not all of which have capacity to

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provide disease control. In certain instances, treatments may minimize symptom expression in

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a temporal fashion yet fail to limit pathogen activity or persistence. For example, independent

107

use of models, including large volume compost applications (Noble and Coventry, 2005) or

108

elevated nutrient inputs (Slykhuis and Li, 1985), lack potential as a soil fumigation replacement

109

as the methods temporally alleviate plant nutrient deficiencies resulting from loss of root

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function due to pathogen activity but fail to address disease causality. In contrast, mustard seed

7 111

meal (MSM) soil amendments or anaerobic soil disinfestation (ASD) are methods that improve

112

tree growth both through enhanced soil fertility (Snyder et al., 2009; Di Gioia et al. 2017) and

113

pathogen suppression (Mazzola et al., 2007; Momma et al., 2013). Disease control attained in

114

response to either method involves a complex of often inter-dependent biological and chemical

115

mechanisms (Angus et al., 1994; Hewavitharana et al., 2014; Rosskopf et al., 2015), and may

116

require function of specific components of the soil microbiome to obtain optimal disease

117

control activity (Cohen et al. 2005; Weerakoon et al., 2012; Mowlick et al., 2013;

118

Hewavitharana and Mazzola, 2016b). Both ASD and MSM soil amendment have yielded

119

effective control of apple replant disease in field trials at a level equivalent or superior to that

120

attained through soil fumigation (Mazzola et al., 2015; Hewavitharana and Mazzola, 2016a;

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Wang and Mazzola, 2019a).

122

Adoption of ASD and MSM as soil-borne disease management strategies has been impeded

123

by input product expenses, particularly with respect to the use of the effective seed meal

124

formulation (Mazzola et al., 2015), which possess an upfront cost triple that of pre-plant soil

125

fumigation. Integration of ASD and MSM amendment may result in a reduced cost disease

126

control strategy, while also expanding or extending the period of pathogen suppression. Such

127

an outcome could stimulate grower adoption of these alternatives if demonstrated to

128

consistently outperform fumigation in terms of yield on replant sites, which has been observed

129

in field trials conducted with Brassica juncea:Sinapis alba (1:1) MSM soil amendment (Mazzola

130

et al., 2015; Wang and Mazzola, 2019a). As a functional microbiome is vital to the disease

131

control capacity of ASD or MSM amendment, co-application or sequential application has

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potential to alter treatment efficacy due to unanticipated or unknown effects on soil biology.

8 133

Incorporation of B. juncea seed meal to moist soil results in generation of the biologically active

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volatile AITC which functions directly and indirectly in the suppression of soil-borne plant

135

pathogens (Cohen and Mazzola 2006, Weerakoon et al. 2012). Generation of seed meal

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derived AITC may be altered by application sequence due to physical or biological modifications

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to the soil system. Therefore, studies were conducted to assess whether sequential application

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of ASD and MSM soil amendment influenced i.) generation of active chemistries, ii.)

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composition of the soil microbiome and iii.) root infection by replant disease pathogens.

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2. Materials and Methods

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2.1 Orchard Soil

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Studies were conducted using soil obtained from a commercial (GC) apple orchard located

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near Manson, Washington, USA (latitude 47°53ˊ05 ̋N, longitude 120°09 ˊ30 ̋ W). The orchard

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was established in 1991 on ground that was not fumigated prior to re-planting the site with

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‘Golden Delicious’ on M.7 rootstock. The pathogen complex contributing to replant disease at

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this site includes various species of Pythium, Phytophthora cactorum, Rhizoctonia solani

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anastomosis groups (AG) 5 and 6, Ilyonectria robusta and Pratylenchus penetrans. The

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dominant soil type at this orchard is a Chelan gravelly sandy loam, with a pH of 6.0 and organic

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matter content of 3.2%. Soil was collected from the root zone of multiple randomly selected

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trees at a depth of 10-30 cm and transported to the USDA-ARS Tree Fruit Research Laboratory,

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Wenatchee, WA. Prior to use, soils were mixed using a cement mixer to obtain a representative

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orchard soil sample. Soil was collected in October 2013, May 2015 and October 2016 for use in

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experiments 1, 2 and 3, respectively.

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2.2 Soil treatments

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Soil treatments used in this study included anaerobic soil disinfestation with grass as the

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carbon input (ASD-GR) at 10 t ha-1, ASD with rice bran as the carbon input (ASD-RB) at 10 t ha-1,

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MSM composed of a 1:1 formulation (Mazzola et al., 2015) of Brassica juncea cv. Pacific Gold

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and Sinapis alba cv. IdaGold applied to soil at a rate of 4.4 t ha1, soil pasteurization and a no

159

treatment control. Furthermore, ASD and MSM were included in sequential combinations of

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ASD-GR or ASD-RB treatment followed by MSM soil amendment and conversely, MSM soil

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amendment followed by ASD-GR or ASD-RB application.

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2.3 Application of soil treatments

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2.3.1 Anaerobic soil disinfestation

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A mixed grass sample composed primarily of Poa pratensis, Poa bulbosa, Bromus tectorum

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and Bromus inermis (2.99% N, 0.37% P, 2.50% K; Soiltest Farm Consultants, Moses Lake, WA)

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was cut and collected from the Columbia View Research and Demonstration (CV) orchard near

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Orondo, Washington, USA. The grass was air dried for three days on a greenhouse bench and

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cut into segments approximately 1-cm in length prior to use. Rice bran (2.39% N, 1.08% P,

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1.54% K; Soiltest Farm Consultants) used in these trials was obtained from Farm Fuels Inc.

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(Santa Cruz, CA). Orchard soil (800 g) was placed into 12.5 cm dia plastic pots and the

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appropriate carbon source was mixed thoroughly into soil at the rates stated above. Soils were

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then watered to field capacity and all pots were sealed in a double layer of two gas

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impermeable transparent Saranex bags (17.8 × 20.3 cm, Bitran Series “S”bags, Com–Pac

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International Carbondale, IL). Pots were incubated in environmental growth chambers using a

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day/night temperature regime of 24/18 °C with a 12 h photoperiod over a period of one week.

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Pots were then removed from bags and soils were aerated for a period of three (experiment 1)

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or four (experiments 2 and 3) weeks.

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2.3.2 Mustard seed meal soil amendment

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The MSM formulation (3.48% N, 0.59% P, 0.70% K) was produced by blending the individual

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B. juncea and S. alba derived seed meals together (1:1), grinding the flaked meal fragments

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with a blender, and passing the resulting particles through a 1-mm2 sieve prior to application.

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MSM was thoroughly incorporated into soil by hand and 800 g of treated soil was placed into

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an individual pot. Each pot was sealed in Saranex bags and incubated in environmental growth

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chambers under the conditions described above. Sealed bags were opened after completion of

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allyl isothiocyanate (AITC) generation in the MSM treated soil (72 h; Mazzola et al., 2007) and

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soils were aerated as noted above for ASD. At completion of the post-ASD and MSM treatment

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aeration period, soils either received the alternate treatment of ASD or MSM, or were directly

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planted with apple seedlings. Application of treatments was coordinated in such a temporal

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manner that resulted in termination of all required aeration periods at the same time for all

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treatments allowing for use of a single uniform planting date. For soil pasteurization,

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moistened soil was placed in a plastic bag and heated overnight at 70° C on two successive

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days. Each treatment consisted of 5 replicates and pots were arranged in a complete

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randomized design on a greenhouse bench.

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2.4 Assessment of AITC production as affected by prior ASD treatment

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Assessment of AITC production was evaluated in experiments discrete from the plant

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bioassays described below. ASD treatments using grass or rice bran as the carbon input were

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applied to potted soils as described above. The MSM formulation was incorporated into ASD-

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GR, ASD-RB or non-treated orchard soil as described above and the trial included a no MSM

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amendment control for analysis of AITC. Potted soils were immediately sealed in Saranex bags

200

and after three hours incubation, a volatile sample was collected from the headspace of the

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bagged pot by purging air into a Tenax trap (60-80 mesh) porous polymer in a silanized glass

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tube. The air was purged using a Magnatek pump (Universal Electric Motor 115 V/ 60 Hz). The

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purge flow time to collect a specified volume of air was established using a glass flowmeter

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system, as described by Hewavitharana et al. (2014).

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Gas samples were injected into a gas chromatograph with flame ionization detector (GC-FID),

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in which AITC concentrations were calculated from peak area according to injections of an

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authentic standard. The inlet temperature was 250 °C and run in splitless mode. The column

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was a HP-PLOT poraPLOT Q (Agilent 19091P-Q01) (10 m length, 320 µm diameter, 10 µm film

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thickness) (Agilent Technologies, Santa Clara, CA), and the oven program was 175 °C to 200 °C

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at a rate of 35 °C min-1. The GC-FID detector was maintained at 250 °C, with a hydrogen flow

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rate of 25 mL min, air flow rate of 300 mL min, and nitrogen as the makeup gas at 25 mL min.

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2.5 Plant bioassays

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Plant bioassays were conducted with ‘Gala’ apple seedlings which were prepared as

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previously described by Hewavitharana and Mazzola (2016) using seed extracted from apple

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harvested at a 16-year-old planting of Gala/M.26 at the CV orchard. Five 8-week-old apple

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seedlings were transplanted into each pot and plants grown for a period of eight weeks at 22° C

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+ 4° C with a 14 h photoperiod. At harvest, seedling root systems were washed under a stream

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of tap water and plant height, shoot and root fresh weights were determined. A bulk 0.5 g fine

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root sample was collected for each pot for nematode extraction. The root sample was placed in

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80 ml of sterile deionized water in 125-ml flasks and placed on a reciprocal shaker at 140 rpm

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for 6 days. Nematodes were collected by passing the suspension twice through a 37 μm sieve

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and backwashing into a counting dish. Pratylenchus penetrans were identified based upon

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morphological features and counted using a light microscope (×40). For experiments 1 and 2,

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determination of apple root infection by Pythium spp. and Rhizoctonia spp. was conducted by

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plating 20 root segments from each root sample (0.5 to 1.0 cm in length) onto PSSM media

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(Mazzola et al., 2001) and water agar amended with ampicillin (100 μg ml-1), respectively, as

227

described previously (Mazzola, 1998). Identification of these organisms was based upon

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morphological features with additional confirmation of identity obtained via sequencing of the

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internal transcribed spacer (ITS) region of the rRNA genes as described previously (Mazzola,

230

1997; Mazzola et al., 2002)

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For experiment 3, root infestation by P. ultimum, the dominant species of Pythium recovered

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from roots of seedlings grown in this orchard soil during experiment 2, and Rhizoctonia solani

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was determined by quantitative PCR (qPCR). DNA was extracted from a pooled 0.5 mg root

234

tissue sample from each pot using Powerplant Pro® plant DNA isolation kit (MO BIO Inc.). qPCR

235

was conducted using the primer pair ULT 1F/ULT 4R (Schroeder et al. 2006) and Rhsp1/ITS4B

236

(Bruns et al., 1991; Gardes and Bruns, 1993; Salazar et al., 2000) for quantification of P. ultimum

237

and R. solani, respectively. The reaction mixture consisted of 1.0 μl of a 1:100 dilution of root

238

DNA extract, 0.1 μl of each primer (100 pmole μl-1), 3.0 μl SYBR Green PCR Master Mix (Applied

239

Biosystems, Warrington, UK), and 5.8 μl nuclease-free water (Ambion®, Life Technologies,

240

Carlsbad, CA). Standard curves were prepared using purified DNA from P. ultimum (isolate 60-

241

1205) and R. solani AG-5 (isolate 5-103) diluted from 0.1 ng μl-1 to 10 fg μl-1. qPCR was

13 242

conducted using a StepOnePlus Real Time PCR System (Applied Biosystems, Foster City, CA)

243

with three technical replicates for each root sample and the no-template control. Amplification

244

of P. ultimum was conducted using the following conditions: 95 °C 10 min, (95 °C 15 sec-62 °C 1

245

min-72 °C 30 sec) x 40 cycles, followed by a melt curve with a 0.3 °C sec-1 increase in

246

temperature from 60 °C to 95 °C. Reaction conditions for R. solani were as follows: 95 °C 10

247

min, (95 °C 15 sec- 59 °C 30 sec -72 °C 1.5 min) x 40 cycles, with the melt curve generated using

248

the same conditions as described above.

249

2.6 Microbial community analysis

250

The effect of ASD and MSM treatments on soil and rhizosphere microbial community profiles

251

was examined in experiment 3. A 5-g soil sample was collected from each pot immediately

252

prior to apple seedling planting and a 5-g rhizosphere soil sample was collected from each pot

253

at harvest, eight weeks post-planting. DNA was extracted from the entire 5-g soil samples using

254

the DNeasy PowerMax soil DNA isolation kit (Qiagen, Germantown, MD). Terminal-restriction

255

fragment length polymorphism (T-RFLP) analysis was utilized to profile bacterial and fungal

256

communities in bulk soil (collected prior to planting) and rhizosphere soil (collected at seedling

257

harvest). Fluorescently labeled PCR products of the fungal ITS region were generated using the

258

D4 labeled ITS-1F primer in conjunction with D3 labeled ITS4 primer. D4 labeled 8F and D3

259

labeled 907R primers were used in amplification of the bacterial 16S rRNA gene. Fungal

260

amplicons were double digested using Hae III and Hha I, and bacterial amplicons were digested

261

using Hae III in reaction mixtures containing CutSmart buffer (Biolabs Inc., New England) and

262

nuclease-free water (Ambion®, Life Technologies, Carlsbad, CA). Separation of fragments using

14 263

the CEQ 8000 Genetic Analysis System (Beckman-Coulter, Brea, CA) was conducted as described

264

by Weerakoon et al. (2012).

265 266

2.7 Data analysis

267

Statistical analyses were completed using SAS 9.4 software (SAS Institute Inc., Cary, NC).

268

Similarity in microbial community composition among soil treatment groups was assessed by

269

non-metric multidimensional scaling (NMDS) of bacterial and fungal T-RFLP data using PAST

270

software package ver 3.16 (Hammer et al., 2001). Dice similarity coefficient was calculated

271

among groups of samples and used to perform ordination and one-way analysis of similarity

272

(ANOSIM). Results with P ≤ 0.05 were regarded as significant, with a large positive R (up to 1)

273

signifying dissimilarity among groups (Hammer et al., 2001). Quantitative data such as shoot

274

biomass were analyzed by ANOVA using Proc Mixed or Proc GLM procedures with appropriate

275

data transformations to satisfy the model assumptions. Nematode count data were log10 (count

276

+1) transformed and percentage root infection data were arc sin transformed prior to analysis.

277 278

3. RESULTS

279

3.1 Effect of prior ASD treatment on seed meal derived AITC emission

280

Air samples were collected from the head space of bags containing potted soils at three

281

hours after application of MSM. The sampling point was selected as it corresponds with the

282

timing of peak AITC generation from MSM amended soil under the experimental conditions

283

utilized (Mazzola and Zhao, 2010; Wang and Mazzola, 2019b). Soils that had received ASD-RB

284

treatment prior to incorporation of MSM generally exhibited a lower yield of AITC relative to

15 285

that attained when MSM was applied to untreated orchard soil (Table 1). When MSM was

286

applied to ASD-GR treated soils, AITC levels were either similar to or lower than that generated

287

when seed meal was applied to the untreated soil.

288 289

Table 1 Effect of prior anaerobic soil disinfestation (ASD) treatment using grass (ASD-GR) or rice bran

290

(ASD-RB) on concentration of allyl isothiocyanate (µg ml-1) detected in the head space of sealed gas-

291

impermeable bags containing soils amended with a 1:1 formulation of Brassica juncea:Sinapis alba seed

292

meal (MSM).

Allyl isothiocyanate (µg ml-1 head space sample) Soil treatment

Trial 1

Trial 2

0z

0

MSM

2.62 + 0.78

2.47 + 0.06

ASD-GR

2.45 + 0.41

0.83 + 0.03

ASD-RB

0.47 + 0.10

1.10 + 0.18

Control

293

z

294

average of two gas sample injections assessed by gas chromatography with flame ionization detector.

Values are means of three replicates + standard deviation with each observation determined as an

295 296

3.2 Effect of soil treatments on soil microbiome

297

NMDS ordination of T-RFLP derived data as assessed in experiment 3 indicated significant

298

effect of soil treatment on structure of the bulk soil bacterial and fungal community (Fig. 1).

299

Fungal community composition for all treated soils differed significantly (P < 0.009) from the

300

control at planting with pairwise RANOSIM values as high as 1.0 (Table 2). There were no

301

significant (P > 0.439) differences in soil fungal community composition among treatments that

302

possessed ASD-RB as a factor. The fungal community from all ASD-GR treated soils differed

16 303

significantly from that detected in soils receiving ASD-RB treatment or the MSM treatment

304

independently (P < 0.0463). In contrast to the integration of ASD-RB and MSM treatments,

305

sequence of application did significantly affect the fungal community composition when ASD-

306

GR and MSM applications were integrated. Fungal communities from bulk soils receiving the

307

ASD-GR/MSM and MSM/ASD-GR treatments were highly dissimilar (P = 0.008; RANOSIM = 0.912).

308

309 310

Fig. 1. Influence of treatment and application sequence on GC orchard soil fungal (left) and bacterial

311

(right) community composition at planting, as examined in experiment 3 at completion of the four-week

312

post-treatment soil aeration period. Ordination of soil microbiomes was conducted by nonmetric

313

multidimensional scaling of terminal restriction fragment length polymorphism-derived data and

314

distance was based on Dice similarity coefficient. ASD-RB = anaerobic soil disinfestation conducted using

315

rice bran; ASD-GR = ASD conducted using grass; MSM = Brassica juncea: Sinapis alba seed meal

316

formulation (1:1).

317 318

17 319

Table 2 Analysis of similarity R values in pairwise comparisons of fungal community structure in GC

320

orchard replant soil four weeks after application of a Brassicaceae seed meal formulation, anaerobic soil

321

disinfestation (ASD) with grass as the carbon input and ASD with rice bran as the carbon input utilized

322

independently or as integrated soil treatments. Fungal community Soil treatmentz

Control

MSM

ASD-RB

ASD-GR

ASD-GR/ MSM

MSM/ ASD-GR

ASD-RB/ MSM

Control MSM

0.608y

ASD-RB

0.396

0.024

ASD-GR

1.0

0.604

0.388

ASD-GR/MSM

0.924

0.412

0.396

0.868

MSM/ASD-GR

1.0

0.608

0.400

1.0

0.912

ASD-RB/MSM

0.468

-0.060

-0.060

0.408

0.424

0.400

MSM/ASD-RB

0.472

-0.060

-0.244

0.364

0.344

0.544

-0.016

323

z

324

MSM = Brassica juncea: Sinapis alba seed meal formulation (1:1). Sequence of treatment application is

325

indicated by acronym order separated by backslash.

326

y

327

spacer region. R values <0.25 indicate two communities are barely separable; R values between 0.25 and

328

0.5 are considered distinct with some overlap; R values > 0.75 are considered well separated.

329 330 331

ASD-RB = anaerobic soil disinfestation conducted using rice bran; ASD-GR = ASD conducted using grass;

Fungal communities were characterized by TRFLP analysis of the fungal rDNA internal transcribed

18 332

Analysis of similarity indicated that structure of the soil bacterial communities was dissimilar

333

among treatments at the time of planting (P < 0.001; RANOSIM = 0.706). Pairwise comparison of

334

T-RFLP data indicated that bacterial community structure was significantly (P < 0.033) different

335

among all treatments at planting. While most treatments exhibited clear separation in soil

336

bacterial community structure based upon analysis of similarity RANOSIM values (Table 3),

337

pairwise comparison of MSM vs. ASD-RB (R = 0.341) and MSM vs ASD-RB/MSM (R = 0. 456)

338

indicated separation but some overlap in community structure (Ramette, 2007) between the

339

contrasted soils. Bacterial community structure in the control soil was well separated from that

340

detected in all treated soils at planting (R = 0.78 to 1.0). Treatment application sequence

341

significantly affected soil bacterial community structure regardless of the ASD carbon source

342

utilized.

343

19 344

Table 3 Analysis of similarity R values in comparison of bacterial community structure in GC orchard

345

replant soil four weeks after application of a Brassicaceae seed meal formulation, anaerobic soil

346

disinfestation (ASD) with grass as the carbon input and ASD with rice bran as the carbon input utilized

347

independently or as integrated soil treatments Bacterial community Soil treatmentz

Control

MSM

ASD-RB

ASD-GR

ASD-GR/ MSM

MSM/ ASD-GR

ASD-RB/ MSM

Control MSM

0.944y

ASD-RB

0.780

0.341

ASD-GR

0.960

0.794

0.732

ASD-GR/MSM

1.000

0.656

0.676

0.820

MSM/ASD-GR

0.852

0.613

0.648

0.776

0.916

ASD-RB/MSM

0.980

0.456

0.568

0.932

0.544

0.736

MSM/ASD-RB

1.000

0.975

0.792

1.000

0.972

0.888

0.718

348

z

349

MSM = Brassica juncea: Sinapis alba seed meal formulation (1:1). Sequence of treatment application is

350

indicated by acronym order separated by backslash.

351

y

352

<0.25 indicate two communities are barely separable; R values between 0.25 and 0.5 are considered

353

distinct with some overlap; R values > 0.75 are considered well separated.

354 355

ASD-RB = anaerobic soil disinfestation conducted using rice bran; ASD-GR = ASD conducted using grass;

Bacterial communities were characterized by TRFLP analysis of the bacterial 16S rRNA gene. R values

20 356

At harvest, there were few treatment effects on structure of the rhizosphere bacterial

357

community (Fig. 2) with only the ASD-GR/MSM (P = 0.0077) and MSM/ASD-RB (P = 0.0076)

358

differing significantly from the control. However, all pairwise comparisons, between control

359

and treated soils had RANOSIM values of less than 0.25 indicating that the two groups of

360

communities possessed considerable or complete overlap in rhizosphere bacterial community

361

composition (Ramette, 2007).

362 363

Fig. 2. Influence of soil treatment and application sequence on ‘Gala’ apple seedling rhizosphere fungal

364

(left) and bacterial (right) community composition at plant harvest as examined in experiment 3, eight

365

weeks post-planting in GC orchard replant soil. Ordination of soil microbiomes was conducted by

366

nonmetric multidimensional scaling of terminal restriction fragment length polymorphism-derived data

367

and distance was based on Dice similarity coefficient. ASD-RB = anaerobic soil disinfestation conducted

368

using rice bran; ASD-GR = ASD conducted using grass; MSM = Brassica juncea: Sinapis alba seed meal

369

formulation (1:1).

370

21 371

Significant differences in rhizosphere fungal community structure (Fig. 2) were detected at

372

harvest among soil treatments (RANOSIM = 0.6222; P = 0.0001). RANOSIM for pairwise analyses

373

between different groups are shown in Table 4. Significant differences were detected between

374

all treatments (P < 0.0479) with exception of the comparison between ASD-GR and ASD-

375

GR/MSM (P = 0.0594) treated soils. All treated soils possessed a rhizosphere fungal community

376

structure that was dissimilar from that detected in the no treatment control (P < 0.0095). Pair-

377

wise comparison indicated that the rhizosphere fungal community detected in the ASD-GR

378

treatment was most dissimilar from the control (RANOSIM = 1.0). In relation to the control, fungal

379

communities from all soil treatments, with exception of ASD-RB, had pairwise comparison R-

380

values indicating that the two groups of fungal communities were largely or almost entirely

381

distinct (Table 4).

382

22 383

Table 4. Analysis of similarity R values in comparison of ‘Gala’ apple seedling rhizosphere fungal

384

community structure after eight weeks growth in GC orchard replant soil. Soils received application of a

385

Brassicaceae seed meal formulation, anaerobic soil disinfestation (ASD) with grass as the carbon input

386

and ASD with rice bran as the carbon input independently or as integrated soil treatments prior to

387

planting. Fungal community Soil treatmentz

Control

MSM

ASD-RB

ASD-GR

ASD-GR/ MSM

MSM/ ASD-GR

ASD-RB/ MSM

Control MSM

0.780y

ASD-RB

0.482

0.608

ASD-GR

1.0

0.976

0.916

ASD-GR/MSM

0.632

0.248

0.326

0.484

MSM/ASD-GR

0.792

0.472

0.564

0.768

0.320

ASD-RB/MSM

0.784

0.704

0.460

0.912

0.484

0.688

MSM/ASD-RB

0.804

0.340

0.640

0.892

0.308

0.444

0.568

388

z

389

MSM = Brassica juncea: Sinapis alba seed meal formulation (1:1). . Sequence of treatment application is

390

indicated by acronym order separated by backslash.

391

y

392

transcribed spacer region. R values <0.25 indicate two communities are barely separable; R values

393

between 0.25 and 0.5 are considered distinct with some overlap; R values > 0.75 are considered well

394

separated

395 396

ASD-RB = anaerobic soil disinfestation conducted using rice bran; ASD-GR = ASD conducted using grass;

Rhizosphere fungal communities were characterized by TRFLP analysis of the fungal rDNA internal

23 397

3.3 Apple seedling growth

398

At harvest, one or more growth parameters was elevated for apple seedlings cultivated in

399

orchard replant soil treated with ASD or MSM prior to planting relative to the control. For all

400

growth parameters (shoot length, shoot weight, root weight), a significant effect of experiment

401

(P < 0.001) and a significant interaction between experiment and treatment (P < 0.001) was

402

observed. Therefore, seedling growth data are presented by individual experiment (Figs. 3-5).

403

Seedling growth in pasteurized soil consistently was greater than that attained in the control

404

soil indicating the presence of a growth limiting soil-borne pathogen complex. In all

405

experiments, seedling growth attained in response to one or more ASD/MSM treatments was

406

significantly greater than that achieved when seedlings were grown in pasteurized soil.

407

Combining ASD treatment with MSM soil amendment did not reliably yield an improvement in

408

seedling growth beyond that attained with one or more stand-alone treatments, either ASD or

409

MSM. When used independently, seedling growth commonly was similar between ASD-RB,

410

ASD-GR and MSM treated soils, but significantly greater than that attained for seedlings

411

cultivated in the no treatment control soil. However, the ASD-GR treatment demonstrated

412

inconsistency in this response relative to the other soil treatments, and in experiment 3 root

413

biomass of apple seedlings grown in the ASD-GR treated soil was significantly less than that of

414

seedlings cultivated in either ASD-RB or MSM treated soils (Fig. 4).

415

When the integrated ASD/seed meal treatments were applied, a significant effect of

416

application sequence on plant growth was realized when grass, but not rice bran, was used as

417

the ASD carbon input. Application of ASD-GR followed by MSM soil amendment resulted in a

418

superior plant growth response relative to the MSM/ASD-GR application sequence in all three

24 419

experiments. In experiment 1, plants cultivated in the MSM/ASD-GR treated soil exhibited

420

symptoms of phytotoxicity at 4 days post-planting. This observation resulted in extension of

421

the soil aeration period from 3 to a duration of 4 weeks in the subsequent experiments. The

422

ASD-GR/MSM treatment sequence at times was superior to the ASD-GR treatment applied

423

independently in terms of promoting shoot biomass (Fig. 5), but the response was observed

424

only in experiments 2 and 3. In contrast, when MSM amendment preceded ASD-GR in the

425

application sequence, plant growth was typically reduced relative to other ASD or MSM

426

treatments and in experiment 1 the MSM/ASD-GR application sequence did not significantly

427

improve root biomass relative to the no treatment control (Fig. 4).

25

428 429

Fig. 3. Effect of soil treatment on shoot length of ‘Gala’ apple seedlings in GC orchard replant soil. ASD-

430

RB = anaerobic soil disinfestation conducted using rice bran; ASD-GR = ASD conducted using grass; MSM

431

= Brassica juncea: Sinapis alba seed meal formulation (1:1). Sequence of treatment application is

432

indicated by acronym order; e.g. MSM ASD-RB = mustard seed meal amendment followed by anaerobic

433

soil disinfestation conducted using rice bran. Top panel = experiment 1; middle panel = experiment 2;

434

bottom panel = experiment 3. Within a given experiment, means, represented by bars, designated with

435

the same letter are not significantly (P > 0.05) different. Bars indicate standard error of the mean.

26

436 437

Fig. 4. Effect of soil treatment on root weight of ‘Gala’ apple seedlings in GC orchard replant soil. ASD-

438

RB = anaerobic soil disinfestation conducted using rice bran; ASD-GR = ASD conducted using grass; MSM

439

= Brassica juncea: Sinapis alba seed meal formulation (1:1). Sequence of treatment application is

440

indicated by acronym order; e.g. MSM ASD-RB = mustard seed meal amendment followed by anaerobic

441

soil disinfestation conducted using rice bran. Top panel = experiment 1; middle panel = experiment 2;

442

bottom panel = experiment 3. Within a given experiment, means, represented by bars, designated with

443

the same letter are not significantly (P > 0.05) different. Bars indicate standard error of the mean.

27 444

445 446

Fig. 5. Effect of soil treatment on shoot weight of ‘Gala’ apple seedlings in GC orchard replant soil. ASD-

447

RB = anaerobic soil disinfestation conducted using rice bran; ASD-GR = ASD conducted using grass; MSM

448

= Brassica juncea: Sinapis alba seed meal formulation (1:1). Sequence of treatment application is

449

indicated by acronym order; e.g. MSM ASD-RB = mustard seed meal amendment followed by anaerobic

450

soil disinfestation conducted using rice bran. Top panel = experiment 1; middle panel = experiment 2;

451

bottom panel = experiment 3. Within a given experiment, means, represented by bars, designated with

452

the same letter are not significantly (P > 0.05) different. Bars indicate standard error of the mean.

28 453

3.4 Effect of soil treatment on P. penetrans root density Across the three replicate trials, there was no significant effect of experiment (P =0.207),

454 455

there was no significant interaction between experiment and treatment (P = 0.112) but a

456

significant effect of treatment (P < 0.001) was observed. Therefore, data from the three

457

experiments were pooled and presented in Table 5. All soil treatments significantly suppressed

458

numbers of P. penetrans recovered from ‘Gala’ seedling roots at harvest relative to the no

459

treatment control. Lesion nematode densities in the control treatment ranged from 150 to

460

204 g-1 root across experiments but were not detected in roots of seedlings cultivated in

461

pasteurized soil (Table 5). For all other soil treatments, lesion nematode root densities were

462

comparable to that observed for seedlings cultivated in pasteurized soil and there were no

463

significant differences among treatments.

464

Table 5 Effect of soil treatment on density of Pratylenchus penetrans (number g-1 root) recovered from

465

‘Gala’ apple seedling roots cultivated in GC orchard replant soil.

Soil treatmentz Control Pasteurization MSM ASD-RB ASD-GR MSM/ASD-RB ASD-RB/MSM MSM/ASD-GR ASD-GR/MSM

# P. penetrans g-1 root 136.9by 0.0a 1.3a 0.3a 0.3a 0.0a 0.0a 0.0a 0.3a

466

z

467

conducted using grass; MSM = Brassica juncea: Sinapis alba seed meal formulation (1:1).

468

y

ASD-RB = anaerobic soil disinfestation conducted using rice bran; ASD-GR = ASD

Means followed by the same letter are not significantly (P > 0.05; n = 15) different.

29 469 470 471

3.5 Effect of soil treatment on Pythium and Rhizoctonia root infection Dissimilar effects on incidence of root infection by Pythium spp. and Rhizoctonia spp. were

472

observed for seedlings cultivated in treated soils, and relative differences in root infection

473

frequency were observed between the three experiments. In the no treatment control,

474

Pythium spp. apple seedling root infection was relatively low in experiment 1 (5.0%) and trial 3

475

but high in experiment 2 (38%; Table 6). The converse was observed relative to root infection

476

by R. solani for seedlings planted in the control soil in the three trials; low in experiment 2 and

477

high in experiments 1 and 3. In experiment 1, there were no significant differences in Pythium

478

spp. root infection across all treatments. In experiment 2, incidence of apple seedling root

479

infection by Pythium spp. in the MSM, ASD-GR and MSM/ASD-GR treatments was observed at a

480

level comparable to that of the no treatment control. In contrast, Pythium spp. root infection

481

was not detected for seedlings that were cultivated in ASD-RB, MSM/ASD-RB and ASD-GR/MSM

482

treated soils. In experiment 3, the quantity of P. ultimum DNA detected in seedling root DNA

483

extracts was low for all treatments, however P. ultimum was detected at significantly higher

484

levels in roots of seedlings cultivated in ASD-RB treated soil than that observed for all other

485

treated soils including the control (Table 7).

486

30 487

Table 6 Effect of soil treatment on ‘Gala’ apple seedling root infection (%) by Pythium

488

and Rhizoctonia spp. when cultivated in GC orchard replant soil.

% root infection Pythium spp. Soil treatmentz

Rhizoctonia spp.

Exp. 1

Exp. 2

Exp. 1

Exp. 2

Control

5

38bcy

22bc

9

Pasteurization

0

2a

33bc

4

MSM

12

55c

3a

0

ASD-RB

0

0a

22bc

0

ASD-GR

0

23ab

11ab

2

MSM/ASD-RB

2

0a

17ab

5

ASD-RB/MSM

1

9a

0a

4

MSM/ASD-GR

0

30b

41c

0

ASD-GR/MSM

0

0a

11ab

2

489

z

490

MSM = Brassica juncea: Sinapis alba seed meal formulation (1:1).

491

y

492

significantly (P > 0.05; n = 5) different.

ASD-RB = anaerobic soil disinfestation conducted using rice bran; ASD-GR = ASD conducted using grass;

For each experiment, means in the same column followed by the same letter are not

493 494

A similar level of variation was observed among the repeated experiments in the incidence

495

of R. solani root infection as affected by soil treatment. In experiment 1, seedlings cultivated in

496

soils treated independently with MSM or soils in which MSM was applied after ASD-RB

497

treatment exhibited lower levels of R. solani root infection relative to the control. In contrast,

498

when planted in soils in which MSM application preceded the ASD treatment, R. solani root

499

infection was generally elevated relative to the MSM only treatment and did not differ from

500

that observed in the control soil. In experiment 2, R. solani root infection was low, and although

31 501

incidence of infection was commonly lower for seedlings cultivated in treated soils relative to

502

the control, there were no significant differences among treatments. In experimentl 3, the

503

quantity of R. solani DNA detected in roots was significantly lower for seedlings cultivated in all

504

treated soils relative to the no treatment control and there were no significant differences

505

among MSM and ASD treatments (Table 7).

506 507

Table 7 Effect of soil treatment on quantity of Pythium ultimum and Rhizoctonia solani

508

DNA (pg mg-1 root) detected in ‘Gala’ apple seedling root tissue when cultivated in GC

509

Orchard replant soil.

Density (pg mg-1 root) of pathogen DNA detected Soil treatment

Pythium ultimum

Rhizoctonia solani

Control

0.022a

134.55b

Pasteurization

0.000a

10.03a

MSM

0.018a

4.33a

ASD-RB

1.079b

10.51a

ASD-GR

0.049a

0.00a

MSM/ASD-RB

0.007a

0.00a

ASD-RB/MSM

0.000a

0.00a

MSM/ASD-GR

0.000a

0.20a

ASD-GR/MSM

0.061a

5.51a

510

z

511

conducted using grass; MSM = Brassica juncea: Sinapis alba seed meal formulation (1:1).

512

y

513

(P > 0.05; n = 5) different.

514 515

ASD-RB = anaerobic soil disinfestation conducted using rice bran; ASD-GR = ASD

Means in the same column followed by the same letter are not significantly

32 516 517

4. DISCUSSION Anaerobic soil disinfestation and the formulated Brassicaceae seed meal amendment utilized

518

in this study have independently demonstrated effective control of apple replant disease in

519

multiple controlled environment and orchard field trials (Mazzola et al., 2015; Hewavitharana

520

and Mazzola, 2016a; Wang and Mazzola, 2019a). Studies reported here examined whether

521

synergistic or additive benefits toward disease suppression and enhanced plant growth could

522

be obtained through integration of these disease control strategies. In general, the sequential

523

application of the two treatments did not result in any consistent benefit in terms of overall

524

apple replant disease suppression or seedling growth relative to that attained when the

525

treatments were applied independently across the three conducted experiments.

526

Specific constants and inconsistencies were observed among trials relative to the frequency

527

at which pathogens were detected in roots of apple seedlings cultivated in the replant orchard

528

soil. At harvest, root densities of P. penetrans recovered from seedlings grown in the non-

529

treated replant orchard soil were similar across all three trials. In contrast, relative seedling

530

root infection by Pythium spp. and Rhizoctonia spp. differed between the trials with one or the

531

other pathogen being dominant in any individual trial. Orchard soil used in trials 1 and 3, in

532

which incidence of root infection by Rhizoctonia spp. was higher than that of Pythium spp., was

533

collected in the autumn of 2013 and 2016, respectively. In contrast, soil utilized in trial 2,

534

where Pythium spp. was recovered at greater frequency than Rhizoctonia spp. from seedling

535

roots, was obtained from the same orchard site in spring of 2015. The interaction and seasonal

536

variance in dominance of these two pathogens in roots of host plants, including apple, have

537

been documented previously. Phenological studies of Pythium spp. documented that

33 538

populations peaked during cool months and exhibited a low point during summer months in

539

both cultivated and non-cultivated soils (Ali-Shtayeh et al., 1986) with elevated populations

540

corresponding with periods of cool temperature and high soil moisture conditions (Robertson,

541

1973). Prior orchard field studies also observed higher orchard soil densities and apple root

542

infection by Pythium spp. in spring relative to autumn in a given year (Mazzola et al., 2002) and

543

may have contributed to the differences observed in the current study among the replicated

544

trials. In addition, Pythium and Rhizoctonia spp. demonstrate antagonistic relationships, with

545

enhanced root infection by one genus commonly observed at the expense of the other

546

(Pieczarka and Abawi, 1976; Xi et al., 1996; Mazzola et al., 2002).

547

Seedlings cultivated in soil receiving any of the MSM or ASD treatments used in this study

548

exhibited superior growth in terms of shoot length, shoot biomass and root biomass relative to

549

seedlings cultivated in the non-treated orchard soil. The sole exception was detected in trial 1

550

where root biomass of seedlings grown in the MSM/ASD-GR treated soil was no different from

551

the control. Seedling growth promotion in treated soils was associated with suppression of P.

552

penetrans root density in response to ASD or MSM amendment. In contrast, although plant

553

growth response was generally consistent for a given treatment across trials, these responses

554

did not universally correspond with relative recovery of P. ultimum and Rhizoctonia spp. from

555

seedling roots. This outcome may have been influenced by the diversity of biotic interactions

556

with potential to affect plant growth in replant soils, but which were not assessed in this trial,

557

including interactions between Pythium spp. and Cylindrocarpon spp. (Braun, 1981;

558

Tewoldemedhin et al., 2011). Regardless, certain associations between treatment performance

559

and relative infection by Pythium or Rhizoctonia spp. were observed. For example, the

34 560

diminished root biomass observed for the MSM/ASD-GR treatment in experiment 1 was

561

associated with elevated Rhizoctonia spp. root infection. Inferior root biomass detected in the

562

MSM, ASD-GR and MSM/ASD-GR treatments relative to soil pasteurization and ASD-RB

563

treatments in experiment 2 was associated with elevated Pythium spp. root infection. The ASD-

564

RB treatment demonstrated its poorest performance relative to other soil treatments in terms

565

of plant root biomass, in experiment 3. This growth outcome in ASD-RB treated soil was

566

associated with significantly elevated levels of P. ultimum DNA detected in apple seedling roots

567

relative to all other soil treatments.

568

Additional factors, including increased nutrient availability, may have contributed to the

569

overall enhanced seedling growth when cultivated in pasteurized soils or soil treated with ASD

570

or MSM amendment. However, there was no consistent relationship between nutrient inputs

571

attributable to ASD or MSM amendment and corresponding plant growth response. For

572

instance, although the co-application of MSM and ASD-GR would result in the highest N and P

573

input the treatment did not yield the maximum plant growth response. Applied in the

574

MSM/ASD-GR sequence, the treatment resulted in the highest equivalent nutrient input but

575

resulted in plant growth that generally was inferior among soil treatments.

576

Mustard seed meal amendment and ASD provide control of soil-borne plant diseases

577

through a diversity of biological and chemical mechanisms (Cohen et al., 2005; Weerakoon et

578

al., 2012; Momma et al., 2013; Hewavitharana et al., 2014; Runia et al., 2014). The relative

579

function of these diverse mechanisms has potential to be enhanced or diminished when

580

treatments are integrated, which could influence overall disease control efficacy. For instance,

581

the generation of specific chemistries in response to ASD (e.g. dimethyl disulfide) may result in

35 582

enhanced or diminished densities of biological entities (e.g. Trichoderma spp.) that function to

583

suppress pathogen activity in response to seed meal application. Although MSM amendment

584

consistently induces an increase in the abundance of Trichoderma/ Hyprocrea detected in

585

orchard soil (Weerakoon et al., 2012; Wang and Mazzola, 2019a), ASD with grass as the carbon

586

input significantly reduced OTUs representing Trichoderma spp. from 6.26% to 0.05% of the

587

total soil fungal community (Hewavitharana and Mazzola, 2016b). Opposing impacts of the

588

MSM and ASD treatments on specific elements of the soil microbiome that confer pathogen

589

suppression may have contributed to the differential disease control and plant growth

590

responses observed when seedlings were cultivated in ASD-GR/MSM versus MSM/ASD-GR

591

treated soils. The MSM/ASD-GR treatment consistently resulted in inferior disease control and

592

plant growth relative to the ASD-GR/MSM treatment. The fungal communities detected in bulk

593

soil from these two treatments were significantly (P = 0.008) and highly (R = 0.912) dissimilar

594

from each other. While sequence of application generally did not affect treatment efficacy

595

when ASD-RB and MSM amendment were integrated and the bulk soil fungal communities

596

were indistinguishable (R = -0.016) between these treatments, application sequence did yield

597

significant effect on the rhizosphere fungal community at plant harvest. Although these

598

treatments did not reveal benefit of integration for disease control, the potential negative

599

interactions identified based upon application sequence could be of importance in other plant

600

pathosystems or in instances where MSM amendment is utilized as a fertility treatment (Balesh

601

et al., 2005; Lewis et al., 2019), a practice common in organic production.

602

Numerous soil and environmental factors have potential to enhance or limit production of

603

biologically active chemistries in response to application of ASD or MSM amendment (Rosskopf

36 604

et al., 2015; Wang and Mazzola, 2019b). Maximum and total AITC yields obtained over a 9 h

605

post-MSM amendment period from an orchard soil possessing 3.2% OM was only 56.5% of that

606

detected from an orchard soil having an OM content of 1.2% (Wang and Mazzola, 2019b). With

607

a single exception, the yield of allyl isothiocyanate from MSM treated soils was lower when

608

ASD-GR or ASD-RB treatments preceded soil application of MSM. The higher organic matter

609

(OM) possessed in the ASD treated soils, through addition of the grass or rice bran, may have

610

factored into lower AITC levels detected as sorption of isothiocyantes to soil increases with

611

increasing organic matter content (Matthiessen and Shackleton, 2005; Gimsing et al., 2009).

612

The lower AITC levels generated in post-ASD treated soils will have negative effects not only on

613

direct suppression of target pathogens, but also indirect effects by altering successional events

614

in the soil microbiome associated with prolonged disease suppression. For instance, in a

615

separate study (Weerakoon et al., 2012) persistent suppression of root infection by Pythium

616

abappressorium in MSM soil amendment required an intact microbiome and proliferation of

617

Trichoderma spp. in response to AITC generation. When soil AITC concentrations were reduced

618

by allowing for soil aeration rather than tarping the soil to retain the volatile, or by employing

619

large particle sized seed meal, which yields lower levels of AITC, rather than a fine particle seed

620

meal, disease control was compromised (Weerakoon et al., 2012).

621

The findings from this study do not provide justification for integrating ASD and MSM

622

treatments as a means to enhance control of the pathogen complex that incites apple replant

623

disease or promote growth of apple. All individual ASD or MSM treatments performed as well

624

or better than integrated treatments in terms of disease control and apple seedling growth.

625

Similarly, integration of the ASD-RB treatment with MSM amendment did not enhance control

37 626

of Macrophomina crown rot of strawberry relative to ASD-RB treatment alone, and did not

627

improve fruit yields (Muramoto et al., 2016). The lack of additive or synergistic effect upon

628

treatment integration may have resulted from direct negative effects on generation of

629

biologically-active chemistries or indirectly through the inhibition of microbial elements known

630

to function in ASD or MSM-induced disease suppression.

631

Alternatively, elevated levels or interactions among potentially phytotoxic chemistries may

632

have reduced plant growth when the treatments were applied in an integrated fashion. Such

633

an outcome was exhibited in experiment 1 where symptoms of phytotoxicity were observed for

634

seedlings cultivated in the MSM/ASD-GR treatment but a similar response was not observed for

635

the MSM/ASD-RB treatment. These two ASD treatments differ in production of potentially

636

herbicidal chemistries, with ASD-GR resulting in elevated levels and extended persistence of

637

various organic acids and dimethyl disulfide (Klarer and Mazzola, unpublished). Extension of the

638

soil aeration interval between treatment application and planting may minimize the potential

639

for certain of these negative treatment interactions. However, from a management

640

perspective, significant expansion of the post-treatment aeration period will be difficult to

641

incorporate into orchard management programs and is likely to diminish adoption by

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producers. Replant disease control and apple growth response attained in response to ASD or

643

MSM applied independently in this study and observed in multiple field trials (Mazzola et al.,

644

2015; Hewavitharana and Mazzola ,2016a; Wang and Mazzola, 2019a) demonstrate that these

645

measures as currently employed do possess viability as effective alternatives to pre-plant soil

646

fumigation.

647

38 648

Acknowledgements

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This work was supported by funds provided to M. Mazzola through the National Institute of

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Food and Agriculture, United States Department of Agriculture, under award number 2017-

651

51181-26832

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Highlights: • • • •

ASD and MSM application sequence influences soil microbiome composition ASD applied prior to MSM reduces allyl isothiocyanate yield in treated soil ASD and MSM applied independently control apple replant pathogens Integration of the two methods does not enhance replant disease control

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: