Targeted Acquisition of Fusarium oxysporum f. sp. niveum Toxin

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Biotechnology and Biological Transformations

Targeted acquisition of Fusarium oxysporum f. sp. niveum toxindeficient mutant and its effects on watermelon Fusarium wilt Xing-Guang Xie, Chun-Yan Huang, Zhen-Dong Cai, Yan Chen, and Chuan-Chao Dai J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02172 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 21, 2019

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Journal of Agricultural and Food Chemistry

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Targeted acquisition of Fusarium oxysporum f. sp.

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niveum toxin-deficient mutant and its effects on

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watermelon Fusarium wilt

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Xing-Guang Xie1,2,a, Chun-Yan Huang1,a, Zhen-Dong Cai1,3, Yan Chen4, Chuan-Chao

5

Dai1*

6

1Jiangsu

Key Laboratory for Microbes and Functional Genomics, Jiangsu Engineering

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and Technology Research Center for Industrialization of Microbial Resources,

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College of Life Sciences, Nanjing Normal University, Nanjing, Jiangsu Province,

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210023, China.

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2National

Key Laboratory of Plant Molecular Genetics, Chinese Academy of Sciences

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Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and

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Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,

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Shanghai 200032, China.

14 15 16

3Key

Laboratory of Animal Protein Deep Processing Technology of Zhejiang

Province, Ningbo University, Ningbo, Zhejiang Province, 315211, China. 4State

Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science,

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Chinese Academy of Sciences, Nanjing, Jiangsu Province, 210008, China.

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Corresponding author: Chuan-Chao Dai

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Address: College of Life Sciences, Nanjing Normal University, No. 1 Wenyuan Road, Nanjing, Jiangsu province, 210023, China.

20 21

E-mail: [email protected]

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Tel: +86 25 85891382

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Fax: +86 25 85891382

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aThese

authors contributed equally to this work

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ACS Paragon Plus Environment


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ABSTRACT

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Watermelon Fusarium wilt is a common soil-borne disease that has significantly

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affected its yield. In this study, fusaric acid-deficient mutant designated as △ FUBT

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(mutated from Fusarium oxysporum f. sp. niveum, FON) was obtained. The △ FUBT

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mutant showed significant decrease in fusaric acid production but maintained wild-

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type characteristics, such as in vitro colony morphology, size and conidiation. A field

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pot experiment demonstrated that △ FUBT could successfully colonize the

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rhizosphere and the roots of watermelon, leading to significant reduction in FON

33

colonization in watermelon plant. In addition, △ FUBT inoculation significantly

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improved the rhizosphere micro-environment and effectively increased the resistance

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in watermelon. This study demonstrated that a non-pathogenic Fusarium mutant ( △

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FUBT) could be developed as an effective microbial control agent to alleviate

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Fusarium wilt disease in watermelon and increase its yield.

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KEYWORDS: Watermelon (Citrullus lanatus L); Soil sickness; Fusarium wilt;

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Nonpathogenic

Fusarium

mutant;

Rhizosphere;

Plant

antioxidant

enzymes

2


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INTRODUCTION

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Watermelon (Citrullus lanatus L.) belongs to Cucurbitaceae family and regarded

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as one of the main horticultural crops worldwide. The global planting area of

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watermelon is approximately 3.5 million hectares, with total production of

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approximately 100 million tons each year.1 However, watermelon is extremely

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susceptible to fungal pathogens resulting into high yield losses, especially in long-

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term continuous cropping soils.2,3 One of these pathogens is Fusarium oxysporum f.

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sp. niveum (FON), which can cause serious soil-borne Fusarium wilt, a phenomenon

48

known as soil sickness.4 The mycelia, chlamydospores and sclerotia of FON can

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overwinter in soil, and the root exudates of watermelon can promote their growth.5,6

50

leading to significant deterioration of soil microbial flora and reduced soil enzyme

51

activities.7 In addition, Fusarium spp. can produce various phytotoxins, including

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fusaric acid that can cause watermelon Fusarium disease.2 Previous studies have also

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reported that fusaric acid can damage host cell membranes, increase reactive oxygen

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content of mitochondria, prevent ATP synthesis, and induce programmed death of

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cells.2,8-10 Therefore, pathogen accumulation in the rhizosphere and imbalance in

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micro-environment can lead to Fusarium wilt of watermelon.

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Several traditional methods have been applied in agricultural cultivation to

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prevent watermelon Fusarium wilt, including grafting techniques, improved

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cultivation systems, suitable intercropping, relay cropping, crop rotation and

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application of fungal pesticides.3,11,12 However, these methods have not achieved

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large-area application because of their inconsistent and unreliable results, further

62

exacerbating the Fusarium wilt.12 In contrast, the use of biocontrol agents, such as

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arbuscular mycorrhizal (AM) fungi, plant growth-promoting bacteria (PGPR) and

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even non-pathogenic pathogens have been considered as environment-friendly, safe, 3



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and cost-effective biological measures for controlling this disease.4,13,14 Moreover,

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these strains exhibited diverse control mechanisms, such as direct competition for

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nutrition with pathogens, reduction in the germination rate of pathogenic spores,

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competition for infection sites on host roots and triggering plant defense responses.15-

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18

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watermelon using beneficial microbial agents, since colonization by these biocontrol

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agents is usually unstable, with negligible ecological effects in watermelon

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rhizosphere.12,19 Thus, it is required to develop a novel strategy to alleviate the

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watermelon cultivation impediments caused by Fusarium wilt.

However, limited studies have addressed the alleviation of Fusarium wilt of

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In a previous study, Freeman and Rodriguez (1993)20 produced a single locus

75

mutated non-pathogenic Colletotrichum magna (Path-1) that could retain the wild-

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type characteristics, such as in vitro sporulation, spore adhesion, appressorium

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development and infection. Path-1 could grow as an endophyte in host tissues, and its

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prior inoculation protected the plants from disease caused by Colletotrichum and

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Fusarium. Other studies reported that the inoculation of non-pathogenic Fusarium

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oxysporum mutant strains could significantly reduce the death of watermelon

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seedlings due to Fusarium wilt under laboratory conditions.4,21,22 These results

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demonstrated that the development of non-pathogenic mutants from wild-type virulent

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strains might be a novel biological strategy for the biocontrol of Fusarium wilt.

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However, the screening process is usually time-consuming, less specific, and thus

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difficult to obtain strains that have practical application in agricultural production.

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Hence, only few studies have reported the utilization of non-pathogenic Fusarium

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mutation strains as beneficial microbial agents to alleviate watermelon Fusarium wilt

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in natural soil environment.

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Fusaric acid is a well-known phytotoxin produced by FON, which is considered 4


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as the main causative agent of Fusarium wilt in watermelon.2 FUBT gene (GenBank

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Accession No. MK411747) plays an essential role in fusaric acid synthesis in

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Fusarium spp. and is located downstream of polyketide synthase gene, encoding an

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important transporter protein belonging to putative major facilitator superfamily.23

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Thus, the acquisition and application of fusaric acid-deficient mutants might be

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beneficial for Fusarium wilt control because this mutant strain that lacks the

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pathogenicity caused by fusaric acid may compete efficiently than other biocontrol

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organisms. In this study, a fusaric acid-deficient mutant ( △ FUBT) was obtained by

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homologous recombination method, which lacked fusaric acid transport function that

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significantly reduced its secretion. The objectives of this study were to investigate the

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potential of △ FUBT and its action mechanisms to alleviate watermelon Fusarium

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wilt disease.

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MATERIALS AND METHODS

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Fungal strains and culture conditions

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Fusarium oxysporum f. sp. niveum (FON) was previously isolated from a

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watermelon seedling with Fusarium wilt (single-spore isolation) at Hengyang Red

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Soil Experiment Station of the Chinese Academy of Agricultural Sciences in Qiyang,

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Hunan Province. The FON was stored at 4°C on potato dextrose agar (PDA, 200 g L−1

108

potato extract, 20 g L−1 glucose, and 20 g L−1 agar, pH 7.0) and cultured in potato

109

dextrose broth (PDB, 200 g L−1 potato extract and 20 g L−1 glucose, pH 7.0) for 3

110

days at 28°C at 170 rpm in an orbital shaker.

111

Construction of plasmid vector and fungal transformation

112

Primers

(Fubt-F

5′AACTGGTCCAGCTTCTGC

3′

and

Fubt-R

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5′ACTGGGATTACCTCCATAGCAA3′) were used to amplify FUBT gene, and the

114

amplified sequence was compared with related sequences from Fusarium genome 5



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database. A highly homologous putative protein supercont1.108 of Fusarium

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oxysporum PHW808 was obtained and its sequence resembled to FUBT gene. We

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further obtained the relatively long upstream (F1) and downstream (F2) sequences of

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FUBT gene from the genome database of F. oxysporum PHW808, which served as the

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templates for amplifying 5′-flanking (F3) and 3′-flanking (F4) sequences of FUBT

120

gene, respectively. In order to construct the FUBT gene replacement vector, 5′-

121

flanking (F3) and 3′-flanking (F4) sequences of FUBT and a hygromycin (hyg)

122

sequence were amplified using P1/P3, P4/P6 and Hyg-F/Hyg-R primers, respectively

123

(Figure 1 and Table 1). Fusion PCR (F3, F4 and Hyg fragments) was performed to

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obtain fusion DNA fragment (F5) and 5′-flanking fragment-hygromycin-3′-flanking

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fragment with P2/P5 primers (Figure 1). The fusion fragment was further ligated with

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a vector using pEASY-Blunt Zero Cloning kit (Beijing Trans Gen Biotech Company)

127

to obtain a transformed plasmid with hygromycin resistance. In addition, the

128

protoplasts of F. oxysporum were produced according to previously described method

129

of Powell and Kistler (1990),24 and fungal transformation experiments were

130

performed by standard polyethylene glycol method as described by Malardier et al.

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(1989).25 Hygromycin resistant transformants were selected on PDA medium

132

containing 100 µg mL-1 of hygromycin B. Stable transformants were observed by

133

PCR (using Hu-F/Hu-R and Fubt-F/Fubt-R primers) to confirm the presence of

134

hygromycin at correct position and the absence of wild type FUBT gene sequence.

135

Analysis of FUBT disruptants (△FUBT)

136

An in vitro dual culture bioassay was performed to investigate whether the

137

FUBT gene deletion could affect FON colony morphology compared to its wild type.

138

△ FUBT and FON were cultured separately on PDA medium for 7 d at 28°C. Fungal

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agar plugs (9 mm) were taken from △ FUBT and FON plates and transferred to the 6


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experimental PDA plates, with two fungal plugs placed 5 cm apart. Colony growth

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was observed after 5 d of incubation. The conidiation and spore morphology of △

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FUBT strain were observed after 10 d of incubation (previous study showed that

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experimental strains can produce conidia on PDA plates).26 A 3-mm fungal agar plug

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was removed from PDA plate and suspended in 1 mL of distilled water. The number

145

of spores in the suspension was counted with a hemocytometer, and spore

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morphology was observed under a light microscope. All plates were incubated at

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28°C and 70% relative humidity. For each experimental treatment, at least ten

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biological replicates were used.

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Further, the concentration of fusaric acid in △ FUBT and FON were determined.

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△ FUBT and FON were inoculated separately into PDB medium and incubated at

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28°C, 170 rpm for 14 d. The culture medium was mixed with an equal volume of

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methyl caproate, shaken for 20 min and allowed to stand for 30 min to separate the

153

organic phase. The organic phase was evaporated to dryness, dissolved in 500 μL of

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chromatographic grade methanol (Hanbon Sci. Tech. Co. Ltd, China) and filtered

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through a 0.22 μm hydrophobic filters before HPLC (high-performance liquid

156

chromatography) analysis. Fusaric acid contents were qualitatively analyzed on an

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Agilent 1290 Infinity HPLC system with UV detector (Agilent, Palo Alto, CA, USA)

158

and Agilent ChemStation Software. The system was equipped with an Agilent C18

159

column (250 mm × 4.6 mm, 5 μm), and methanol and water (85:15, v:v) was used as

160

mobile phase. The flow rate, injection volume, and detection wavelength were set at

161

0.6 mL min−1, 20 μL, and 274 nm, respectively.

162

Determination of △FUBT pathogenicity

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△ FUBT and FON spore suspensions were collected by flooding 10-day-old

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cultures on PDA with sterile distilled water. After centrifugation, spore pellets were 7



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rinsed with distilled water, and spore concentration was adjusted to 103 mL-1 or 2

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×103 mL-1. The watermelon variety “Heimeiren” was used in this study, which is a

167

common cultivar grown in China. Surface-disinfected seeds were pre-germinated in a

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biochemical incubator until the seedlings reached euphyllia stage, and then they were

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transplanted individually into plastic pots (28 cm diameter and 23 cm depth)

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containing sterile vermiculite. Only △FUBT or FON spore suspensions (100 mL, 103

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spores mL-1) were added to the pots for △ FUBT and FON treatments, respectively.

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Both △ FUBT and FON (at 50 mL, 2 × 103 spores mL-1) were inoculated together for

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△ FUBT+FON treatment. Sterilized distilled water (100 mL) was used as control

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(CK). Hoagland’s nutrient solution was added every 2 days, and sterilized distilled

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water was added when required. After 21 d of incubation, disease severity was

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estimated using a scoring system previously described by De Cal et al. (1995)27 as

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follows: 1, healthy plant, all green leaves; 2, lower leaves yellow; 3, lower leaves

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dead and upper leaves yellow; 4, lower leaves dead and upper leaves wilted; and 5,

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dead plant. Thirty seedlings were evaluated in each treatment.

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Pot experimental design and sample collection

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The field pot experiment was conducted at Nanjing Normal University Botanical

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Garden (Jiangsu province, China, 28°13′N, 116°55′E). Experimental red soils (5-20

183

cm depth) were collected from 3 year-old continuously cropped upland watermelon

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field at Red Soil Experiment Station, Chinese Academy of Agricultural Sciences,

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Hunan Province, China (N26°45′, E111°53′). The collected soil was particularly used

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because watermelon in this field exhibited higher incidence (~60-80%) of Fusarium

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wilt compared to the discontinuous monocultured field in the last two years. The soil

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was classified as Ultisol (FAO classification, 1998). After the removal of organic

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debris, the collected soil was divided into two parts for air-drying to determine the 8


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physical and chemical properties and stored at 4°C for further experiments. Basic

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physicochemical properties of soil were determined as follows: organic matter, 11.8 g

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kg−1; total nitrogen, 0.9 g kg−1; total phosphorus, 0.5 g kg−1; total potassium, 13.9 g

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kg−1; available nitrogen, 77.3 mg kg−1; available phosphorus, 25.2 mg kg−1; available

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potassium, 588.3 mg kg−1, and pH 4.8 (1:2.5, w:v).

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Following experimental treatments were performed to examine the effects of △

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FUBT addition on watermelon growth. △ FUBT was first cultured in PDB for 4 d at

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28°C at 220 rpm in an orbital shaker. Fungal mycelia were collected and washed three

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times with distilled water. A portion of mycelia was used to measure dry weight, and

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the remaining was diluted with distilled water and used as △ FUBT inoculants.

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Approximately 15 kg of soil was taken and transferred to a plastic pot (28 cm

201

diameter and 55 cm depth). Each pot was supplemented with 3 g of urea, 6 g of

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calcium magnesium phosphate and 0.75 g of potassium chlorate. These pre-

203

germinated seeds were first placed into cultivation boxes (28 cm length, 18 cm width

204

and 10 cm height) containing sterile vermiculite to cultivate the seedlings. After 10 d

205

of cultivation, seedlings at similar development stages were transplanted into

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experimental pots. In this study, three treatments (CK, AF and SF) were performed,

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each with 40 pots, with one seedling each. In AF treatment, 50 mL of △ FUBT

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inoculum (3.4 mg L-1) was added to each seedling transplanting hole. An equivalent

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amount of sterilized △ FUBT inoculum (121°C, 20 min) was added in SF treatment.

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Non-inoculated seeds (CK) were treated with 50 mL of sterilized distilled water. All

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pots were randomly arranged on the soil 5 cm above the ground, with watering and

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weeding performed as required.

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After transplanting, six randomly selected watermelon plants were collected at

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flowering stage (S1, 35 d post inoculation), fruit setting stage (S2, 55 d post 9



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inoculation) and maturation stage (S3, 80 d post inoculation) to detect FON and △

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FUBT colonization. In addition, physiological indices of the plants were measured.

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Rhizosphere soil was also collected by physically shaking the soil from the roots and

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used to determine △ FUBT and FON colonization, soil microbial community

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composition and enzyme activities. After harvesting, watermelon Fusarium wilt and

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agronomic index were evaluated. Besides, 0 d soil sample (S0, 0-15 cm, after

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fertilization) was collected prior to seedlings transplanting.

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Determination of △FUBT colonization and microbial activity in soil

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FON counts in the rhizosphere were performed using Fusarium-selective

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medium (FSM) as described by Komada (1975)28 (since FON is considered as the

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dominant Fusarium species in experimental red soil, FSM was used to roughly count

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FON in this study). Rhizosphere △ FUBT counts were determined using FSM by

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adding 0.1 g L-1 of hygromycin B. The bacterial counts in the rhizosphere were

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determined using beef extract peptone, the actinomycetes counts were determined

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using Gause's synthetic medium, and the fungal counts were determined by

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Dichloran-Rose Bengal medium.29 In addition, FON and △ FUBT in the watermelon

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roots were isolated and counted.30

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Soil urease activity was determined by sodium phenoxide colorimetric method

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and expressed as mg NH4-N g-1 of soil (37°C, 24 h).31 Similarly, soil sucrase activity

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was determined by 3,5-dinitrosalicylic acid method and expressed as mg glucose g-1

235

of soil (37°C, 24 h).31 Soil polyphenol oxidase activity was determined by pyrogallol

236

colorimetric method and expressed as mg purpurogallin g-1 of soil (30°C, 2 h).32 Soil

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catalase activity was determined using potassium permanganate titration method and

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expressed as 0.002 mol L-1 KMnO4 mL g-1 of soil (30°C, 30 min).31 Soil phosphatase

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activity was determined using 4-nitrophenyl phosphate as an orthophosphate 10


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monoester substrate and expressed as μg 4-nitrophenol g-1 of soil (37°C, 1 h).33 Fresh

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soil was used to analyze these enzyme activities and all values were expressed on an

242

oven-dried soil basis.

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Denaturing gradient gel electrophoresis (DGGE)

244

Bacterial 16S rDNA was amplified using universal forward (357f-GCclamp) and

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reverse primers (517R), while fungal 18S rDNA was amplified using forward (GC-

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Fungclamp) and reverse primers (NS1) (Table 2). Bacterial PCR products were

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analyzed on 8% acrylamide/bis-acrylamide gel with a denaturing gradient of 35-65%

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(100% denaturant contained 7 M urea and 400 g L−1 formamide). Similarly, fungal

249

PCR products were separated on 6% acrylamide/bis-acrylamide gel with a denaturing

250

gradient of 25-45%. After running for 12 h (bacteria) or 10 h (fungi) at 100 V using

251

CBS-DGGE system (CBS Scientific Co., Inc., Del Mar, CA, USA), gels were stained

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with 1:10000 SYBR Green I (Invitrogen Molecular Probes, Eugene, OR, USA) and

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analyzed under GelDOC-ItTS imaging system (Ultra Violet Products, Upland, CA,

254

USA).

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Determination of root vigor and antioxidant enzymes activities

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Root vigor was determined at seedling (S0, 10 d post inoculation), flowering (S1,

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35 d post inoculation), fruit setting (S2, 55 d post inoculation) and maturation (S3, 80

258

d post inoculation) stages according to previously described method by Yuan et al.

259

(2016).34 Fresh watermelon roots (0.5 g) were washed with distilled water and placed

260

in tubes containing 10 mL of 0.4% triphenyltetrazolium chloride and 66 mM

261

phosphate buffer solution (pH 7.0). The mixtures were incubated at 37°C for 3 h, and

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the reaction was stopped by adding 1 M sulfuric acid. Absorbance was determined at

263

485 nm using a spectrophotometer.

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In addition, antioxidant enzymes activities, such as superoxide dismutase (SOD), 11



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peroxidases (POD), catalase (CAT) and malonaldehyde (MDA) were determined

266

using the respective kits procured from Nanjing Jiancheng Bioengineering Institute

267

(Jiangsu province, China).

268

Determination of disease incidence and agronomic indices

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At maturation stage, the Fusarium wilt severity of watermelon was estimated

270

according to the method described above.27 In addition, watermelon agronomic

271

indices, such as root length, vine length, stem diameter, fresh weight, individual fruit

272

weight, and total yield, were determined.

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Statistical analysis

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The results of DGGE profiles were analyzed using Gelcompar II software

275

(Applied Maths, Austin, USA). The Shannon-Weaver index (H) was calculated from

276

H = (ni/N) log (ni/N), where ni is the peak height of a band and N is the sum of all peak

277

heights in a lane. Principal correspondence analysis (PCA) was performed with SPSS

278

13.0 (SPSS Inc., Chicago, IL, USA) for community ordination using relative band

279

intensity data from Gelcompar II analysis. The significance of differences was

280

determined through one-way analysis of variance (ANOVA) tests via Tukey's

281

multiple comparison method or through Student's t test using SPSS 13.0. The

282

comparisons between different treatments were considered significant at p < 0.05.

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RESULTS

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Phenotypic analysis of ∆FUBT

285

The ∆FUBT mutant was obtained through homologous recombination (Figure 2a

286

and 2b). The transformant colonies were villiform, with white colored mycelium and

287

the morphology was similar to that of wild type FON (Figure 2c). Moreover, colony

288

diameter of ∆FUBT was similar to that of FON (approximately 3-4 cm), indicating

289

that the growth of ∆FUBT was not significantly affected by the disruption of FUBT 12


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gene. Figure 2c showed that no clear inhibition zone was observed, indicating that

291

∆FUBT was not sensitive to FON, which efficiently produces fusaric acid. As shown

292

in Figure 2d and 2e, FON spores were elongated, slightly pointed and tapered at both

293

ends, while the shape of ∆FUBT spore was wider and linear. Further, no detectable

294

change in ∆FUBT conidiation was observed when compared to that of FON (Figure

295

2f).

296

Fusaric acid is required for pathogenicity

297

HPLC analysis showed that the production of fusaric acid in ∆FUBT was

298

significantly reduced compared to that of FON (Figure 2g). The pathogenicity test

299

demonstrated that only ∆FUBT inoculation did not affect the incidence of Fusarium

300

wilt when compared to CK treatment (Figure 2h). However, co-inoculation of ∆FUBT

301

and FON (∆FUBT+FON) significantly decreased the incidence rate by 56.1%

302

compared to FON treatment.

303

FON and ∆FUBT colonization in watermelon rhizosphere and roots

304

The inoculation of ∆FUBT significantly decreased the FON amount in

305

rhizosphere by 16.8% and 16.2% at fruit setting and maturation stages, respectively

306

when compared to CK (Figure 3a). In AF treatment, the amount of ∆FUBT increased

307

significantly from flowering to fruit setting stage and then slightly decreased at

308

maturation stage (Figure 3b).

309

In addition, the inoculation of ∆FUBT significantly decreased the FON

310

colonization in watermelon roots from flowering to maturation stages when compared

311

to CK (Figure 3c). However, the colonization of ∆FUBT in AF treatment was

312

relatively high and stable at all stages (Figure 3d).

313

Rhizosphere microbial quantity and enzyme activities

314

The addition of ∆FUBT significantly increased the rhizosphere bacterial amounts 13



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315

by 34.1%, 23.0% and 30.6% at flowering, fruit setting and maturation stages,

316

respectively when compared to CK (Table 3). At maturation stage, the amount of

317

rhizosphere actinomycetes was found to be increased by 39.0%, while the amount of

318

fungi decreased by 24.6% when compared to CK.

319

Soil urease activity was low before transplantation, and rapidly increased to

320

maximum level during flowering stage before gradually decreasing (Figure 4a).

321

Maximum urease activity was observed in AF treatment that increased by 111%,

322

15.2% and 57.1% at the flowering, fruit setting and maturation stages, respectively

323

when compared to CK. Similarly, soil sucrase activity was significantly increased by

324

51.8%, 36.3% and 21.3% at the flowering, fruit setting and maturation stages,

325

respectively in AF treatment (Figure 4b). Figure 4c demonstrated that soil catalase

326

activity increased gradually over the growth period and reached a maximum at

327

maturation stage. This activity in AF treatment was increased by 29.8% and 7.7% at

328

the flowering and fruit setting stages, respectively. Similar trend was observed for soil

329

polyphenol oxidase activity, with gradual increase during the cultivation process

330

(Figure 4d). At flowering, fruit setting and maturation stages, soil polyphenol oxidase

331

activity was significantly increased by 32.5%, 76.2% and 31.9%, respectively when

332

compared to CK. Further, acid phosphatase activity first increased and then decreased

333

during the sampling period, with no significant intergroup differences (Figure 4e).

334

Soil alkaline phosphatase in the AF treatment showed significant difference only at

335

fruit setting stage, which was 8.6% higher than CK (Figure 4f).

336

Effects of ∆FUBT inoculation on the composition of rhizosphere microbial

337

community

338

∆FUBT inoculation significantly influenced the composition of rhizosphere

339

bacterial community from the flowering to maturation stages (Figure 5a-c). Cluster 14


Page 15 of 44


340

analysis indicated that AF treatment exhibited low similarity with CK and SF

341

treatments, generating a separate clustering branch. In addition, PCA ordination

342

produced similar results (Figure 5d-f), with AF treatment exhibiting different

343

outcomes from others at the flowering, fruit setting and maturation stages. Moreover,

344

at flowering and maturation stages, the richness and diversity indices of bacterial

345

community were significantly higher than that of CK and SF treatments (Table 4).

346

Further, it was observed that the soil fungal community of SF and CK treatments

347

exhibited high similarity from flowering to maturation stages (Figure 5g-i). However,

348

significant differences were observed between CK and AF treatments, with the fungal

349

community in AF treatment generating a separate clustering branch. The PCA

350

analysis also demonstrated that ∆FUBT inoculation highly affected the fungal

351

community from flowering to maturation stages, with a distinct AF treatment in the

352

PCA plots (Figure 5j-l). Further results indicated that the richness and diversity

353

indices of fungal community were significantly decreased in AF treatment at the

354

maturation stage compared to CK (Table 4).

355

Analysis of watermelon root vigor and antioxidative enzyme activities

356

After ∆FUBT inoculation, the root vigor of watermelon significantly increased

357

by 29.7%, 7.5% and 10% at the flowering, fruit setting and maturation stages,

358

respectively when compared to CK (Figure 6a). In addition, the activities of root

359

SOD, POD and CAT were higher by 5.6%, 6.2% and 7.3%, respectively in AF

360

treatment than those in CK treatment (Figure 6b-d). However, the aboveground MDA

361

activity significantly decreased by 14.6% compared to CK (Figure 6e).

362

Analysis of watermelon disease control and agronomic traits

363

Results indicated that the disease index of watermelon plants in AF treatment

364

was significantly reduced by 31.3% compared to CK (Figure 6f). Further, the root 15



Page 16 of 44

365

length, vine length, stem diameter and aboveground fresh weight of watermelon were

366

found to be increased by 11.8%, 43%, 3.6% and 10.6%, respectively than that of CK

367

plants (Table 5). In addition, it was observed that ∆FUBT inoculation significantly

368

increased the single fruit weight and total yield by 8.9% and 8.3%, respectively

369

compared to CK.

370

DISCUSSION

371

Non-pathogenic Fusarium oxysporum mutants can be used as an effective

372

biological control tool against watermelon soil sickness caused by Fusarium wilt

373

because these mutated strains could retain all wild-type characteristics, except

374

pathogenicity.21,22 Thus, the growth, reproduction and infection traits of wild-type

375

pathogens might be affected when co-existed with these mutants in natural soil.20-22

376

However, only few studies have reported the use of non-pathogenic pathogen mutants

377

to successfully alleviate watermelon soil sickness caused by Fusarium wilt. There

378

have been no studies to investigate the effects of inoculation of these strains on the

379

rhizosphere micro-ecological environment in watermelon continuous cropping

380

conditions. In this study, a Fusarium oxysporum mutant (∆FUBT) was obtained by

381

homologous recombination method. Further analysis showed that the spore formation

382

and colony morphology of ∆FUBT were similar to wild-type FON, which was

383

consistent with the previous study reported by Freeman and Rodriguez (1993).20

384

However, in ∆FUBT strain, the secretion of fusaric acid was reduced, with significant

385

reduction in its pathogenicity when compared to the wild-type FON. This was in

386

contrast with the previous study in which deletion of FUB (fusaric acid biosynthetic)

387

gene did not affect the virulence of F. oxysporum on maize seedlings.35 Thus, it was

388

ascertained that the mutant ∆FUBT satisfied all the requirements of a non-pathogenic

389

pathogen to be considered as a biocontrol strain against Fusarium wilt of watermelon. 16


Page 17 of 44


390

The use of beneficial microbial agents to control Fusarium wilt of watermelon

391

is a novel and essential biocontrol measure with unique advantages compared to

392

traditional methods.4,7,13,14 Stable colonization ability of exogenous beneficial

393

microorganisms could be considered as an important factor in their potential

394

biocontrol functions. It has been reported that the inactivation of rho1::hyg in

395

Fusarium oxysporum f. sp. lycopersici (lacking functional Rho1 protein with no

396

pathogenicity) was essential for increasing F. oxysporum infection in the host,

397

however, its practical application on the biocontrol of Fusarium wilt is yet to be

398

explored.36 In this study, the toxin transport-related gene FUBT was selected. The

399

knockout of this gene did not affect the growth and reproduction of mutant strain

400

(∆FUBT). The abundance of ∆FUBT in rhizosphere dynamically varied and was

401

significantly dependent on sampling time. The increase of ∆FUBT in fruit setting

402

stage might be due to the plant root growth and seasonal environmental

403

improvements, such as increased root surface, root exudates, soil relative humidity

404

and temperature, which are important for the survival and colonization of exogenous

405

inoculated fungi.37 In contrast, FON colonization in the rhizosphere was significantly

406

decreased after ∆FUBT inoculation, indicating the niche competitive ability of

407

∆FUBT than that of its wild-type strain. In addition, it was observed that the

408

application of ∆FUBT strain might reduce the infection rate of wild-type FON,

409

leading to a significant reduction in its colonization ability in watermelon roots.

410

However, detailed action mechanisms of ∆FUBT inoculation need to be further

411

investigated. The possible reasons for FON infection and colonization reduction could

412

be attributed to improved plant growth, increased watermelon defense mechanism and

413

decreased rhizosphere FON concentration as a result of ∆FUBT inoculation. In the

414

present study, decreased colonization of FON in the rhizosphere and roots was found 17



415

Page 18 of 44

to be a direct factor for alleviating the incidence of watermelon Fusarium wilt.

416

It has been well documented that the rhizosphere microbial community is

417

responsible for crop growth, development, soil nutrient cycling and soil-borne disease

418

control.29,38 Previous studies have found that community structures of rhizosphere

419

microorganisms are susceptible and can be significantly affected by the inoculation of

420

exogenous microbial agents.7,12,38 However, few studies investigated the effects of

421

non-pathogenic pathogen mutant inoculation on the rhizosphere microenvironment.

422

Rhizosphere bacteria are generally considered as the bio-indicators of soil health,

423

while fungi are usually considered as pathogens, which may deteriorate the

424

rhizosphere environment.29,31 After ∆FUBT application, the type of continuously

425

cropped watermelon soil changed from fungal type to bacterial type, which could

426

have contributed to increased rhizosphere fertility. In addition, molecular

427

fingerprintings indicated that the inoculation of ∆FUBT significantly affected the

428

bacterial and fungal community structures of watermelon rhizosphere from flowering

429

to maturation stages. Root exudates released into soil played an important role in

430

influencing the growth, reproduction and community composition of rhizosphere

431

microorganisms.29 Thus, the changes in watermelon root exudates due to ∆FUBT

432

colonization can be considered as the main factor that altered the rhizosphere

433

microbial community. Some other action mechanisms that might have altered the

434

rhizosphere microbial community should not be ignored. These mechanisms included

435

the induced plant systemic resistance, improved physical and chemical properties of

436

soil, and directed antagonism or synergistic action with the indigenous

437

microorganisms due to ∆FUBT addition. However, these assumptions require further

438

research to elucidate the mode of actions of microbial community composition

439

responses to ∆FUBT inoculation in watermelon rhizosphere. Previous studies reported 18


Page 19 of 44


440

that rhizosphere-associated microbes exhibited important roles in plant health and

441

soil-borne disease control.39,40 The results of this study, to the best of our knowledge,

442

are the first to demonstrate that a nonpathogenic Fusarium mutant (∆FUBT) could be

443

used to improve the microbial communities in the rhizosphere of continuously

444

cropped watermelon and play an important role in in vitro antagonism of FON to

445

alleviate watermelon Fusarium wilt.

446

Soil enzymes play an important role in rhizosphere biochemical processes, such as

447

organic decomposition and nutrient release and fixation, which serve as key indicators

448

of soil fertility and functional microbial diversity.31,32 The urease and invertase have

449

been considered as the most important enzymes that reflect the conversion rate of soil

450

carbon and nitrogen metabolism.32 Catalase was closely related to soil microbial

451

activity and respiration and reflected the reactive intensity of soil microorganisms.41

452

After ∆FUBT inoculation, urease, invertase and catalase activities were increased in

453

watermelon rhizosphere. This indicated that ∆FUBT inoculation could increase soil

454

fertility and activity. These results were consistent with previous studies,

455

demonstrating that the inoculation with some beneficial microbial agents, such as

456

Serratia marcescens RZ-21, Phomopsis liquidambari B3, Phomopsis liquidambari

457

4.1 and Ceratobasidium stevensii B6 could increase the activities of soil enzymes.31,42-

458

44

459

plants, which is conducive to their healthy growth and effectively increases crop

460

resistance ability against soil-borne disease. In addition, soil polyphenol oxidase has

461

been associated with the conversion of aromatic organic compounds to humus in

462

soil.45 Previous studies have found that the accumulation of phenolic compounds in

463

continuous cropping soil could significantly promote Fusarium spp. growth and

464

increase the incidence of watermelon Fusarium wilt.6,46 It was further reported that

In fact, increased soil enzyme activities can support the absorption of nutrients in

19



Page 20 of 44

465

phenolic compounds significantly increased toxin production and pathogenicity.46 In

466

this study, ∆FUBT inoculation significantly increased the soil polyphenol oxidase

467

activity. Thus, the effective degradation of soil phenolic compounds by polyphenol

468

oxidase after ∆FUBT inoculation can be considered as an important factor to reduce

469

FON colonization and Fusarium wilt incidence. It can be confirmed that the

470

improvement of rhizosphere enzyme activities due to ∆FUBT inoculation might have

471

facilitated to alleviate the disease severity of Fusarium wilt of watermelon.

472

Malondialdehyde (MDA) content has been considered as an important indicator

473

of lipid peroxidation and usually increases when plants are subjected to pathogenic

474

fungi infection.47,48 Plant antioxidant enzymes, such as SOD, POD and CAT are

475

involved in eliminating reactive oxygen species, and previous studies have reported

476

that the activities of defense-related antioxidant enzymes in roots and leaves are

477

directly related to the plant resistance against biotic stress.47,48 In the present study,

478

SOD, POD and CAT activities increased in watermelon plants after inoculation with

479

∆FUBT. This can be attributed to ∆FUBT infection and colonization that induced the

480

activities of antioxidant enzymes and reduced oxidative damage. Similar results were

481

observed in previous studies, when plants were infected by non-pathogenic Fusarium,

482

the activities of plant defense-related enzymes significantly increased over time.15,17

483

In addition, the MDA content in watermelon shoots inoculated with ∆FUBT was

484

found to be much lower than that of control plants. These results demonstrated that

485

∆FUBT used in the present study significantly improved the systemic resistance in

486

watermelon plants and effectively inhibited the invasion of pathogenic pathogens.

487

Root vigor is an essential trait used to reflect the quality and metabolic status of root

488

development to some extent34 that was used to detect the degree of root injury in this

489

study. After the addition of ∆FUBT, watermelon plants exhibited higher root vigor 20


Page 21 of 44


490

than the control, demonstrated by increased root length. These results showed that the

491

inoculation of ∆FUBT could effectively improve watermelon growth and increase its

492

systemic resistance that can facilitate in reducing the incidence of Fusarium wilt.

493

The application of non-pathogenic Fusarium could alleviate Fusarium wilt of

494

host plants.4,21,22 However, reports relating to the reduction of watermelon Fusarium

495

wilt through the inoculation of non-pathogenic Fusarium under soil conditions are

496

scarce. As an endosymbiotic microorganism, ∆FUBT could colonize watermelon

497

roots, which not only affected the infection and colonization of wild type FON but

498

also effectively improved the resistance of watermelon plants. As an environmentally

499

adaptive non-pathogenic Fusarium, the inoculation of ∆FUBT exhibited significant

500

effects on the rhizosphere microbial community, enzyme activities and FON

501

colonization, leading to the reduction in the incidence of Fusarium wilt and increased

502

watermelon yield. In addition, others factors, such as indirect inhibition of FON,

503

increased soil nutrient cycling, and secretion of beneficial secondary metabolites for

504

promoting watermelon growth may be considered as important effects resulting from

505

∆FUBT inoculation. In conclusion, this study demonstrated that a non-pathogenic

506

Fusarium ∆FUBT mutant could be used as an effective microbial agent to alleviate

507

watermelon Fusarium wilt.

508

Acknowledgments

509

We would like to acknowledge the National Key R&D Program of China

510

(2017YFD0800705), the National Natural Science Foundation of China (No.

511

31870478, 41501329) and the project funded by the Priority Academic Program

512

Development (PAPD) of Jiangsu Higher Education Institutions of China. In addition,

513

Xing-Guang Xie would like to thank Ms. Meng-Ying Hu for invaluable support over

514

the past years and wishes Meng-Ying Hu speedy recovery. We also express our 21



515

gratitude to the anonymous reviewers and editorial staff for their time and attention.

516

Author contributions

Page 22 of 44

517

Chuan-Chao Dai, Xing-Guang Xie and Chun-Yan Huang designed the

518

experiments. Xing-Guang Xie, Chun-Yan Huang, Zhen-Dong Cai and Yan Chen

519

performed the experiments. Xing-Guang Xie and Chun-Yan Huang prepared the

520

manuscript. All authors contributed to data analysis and finalized the manuscript.

521

Conflicts of interest

522

The authors declare that they have no conflicts of interest.

523

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524

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670

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28


Page 29 of 44


Table 1 List of primers Primer

Sequences

P1

ACTGTTGAAGCTGTCATCTGG

P2

CCGCTGAGGTGTCATTGACT

P3

CCGTCCGTCTCTCCGCATGCtgtgaatgggattcg

P4

cactccacatctccactcgTGGGGAAACATATCT

P5

TATGTTCATCTACCAGTCCCCT

P6

ACGGATGTAACAACTTTACGC

Hyg-F

GCATGCGGAGAGACGGACG

Hyg-R

TCGAGTGGAGATGTGGAGTG

Hu-F

CCAGATCAAAGGCCCGAAC

Hu-R

GCGATGAAGTGGGAAAGCTCG

29



Page 30 of 44

Table 2 PCR-DGGE conditions Target

Bacteria

Primers CGCCCGCCGCGCGCGGCGGG 357F-GC CGGGGCGGGGGCAGGGGGGC CTACGGGAGGCAGCAG 517R

Fungi

ATTACCGCGGCTGCTGG

CGCCCGCCGCGCCCCGCGCCC GC-Fung GGCCCGCCGCCGCCCCCGCCC CATTCCCCGTTACCCGTTG NS1

GTAGTCATATGCTTGTCTC

PCR conditions

Polyacrylamide gel (g L-1)

Denaturing gradient (%)

Electrophoresis time (h)

94℃ for 4 min; 20 cycles of 94 ℃ for 1 min, 65 ℃ for 1 min (-0.5℃ for each cycle), 72 ℃ for 1 min, 55℃ for 1 min,72℃ for 1 min; 72℃ for 10 min

80

35-65

12

94℃ for 4 min; 35 cycles of 94℃ for 30 s, 55℃ for 30 s, 72℃ for 1 min; 72℃ for 10 min

70

25-45

10

30


Page 31 of 44


Table 3 The effects of ∆FUBT inoculation on culturable microbes in watermelon rhizosphere Treatment

Bacteria ( × 107 CFU g-1)

Actinomycete ( × 105 CFU g-1)

Fungi ( × 105 CFU g-1)

CK

AF

SF

S0

1.52 ± 0.19a

1.52 ± 0.19a

1.52 ± 0.19a

S1

2.70 ± 0.09a

3.62 ± 0.14b

2.66 ± 0.19a

S2

1.83 ± 0.06a

2.25 ± 0.12b

1.83 ± 0.12a

S3

0.98 ± 0.20ab

1.28 ± 0.10b

0.83 ± 0.11a

S0

1.44 ± 0.12a

1.44 ± 0.12a

1.44 ± 0.12a

S1

1.27 ± 0.15a

1.40 ± 0.02a

1.25 ± 0.11a

S2

0.85 ± 0.11a

1.20 ± 0.17a

0.98 ± 0.23a

S3

0.77 ± 0.05a

1.07 ± 0.15b

0.65 ± 0.13a

S0

2.05 ± 0.12a

2.05 ± 0.12a

2.05 ± 0.12a

S1

2.18 ± 0.16a

1.95 ± 0.23a

2.25 ± 0.13a

S2

1.93 ± 0.15a

1.90 ± 0.1a

2.43 ± 0.21b

S3

2.58 ± 0.13b

2.07 ± 0.15a

2.77 ± 0.10b

Values are presented as means ± SD of three biological replicates. Each replicate represented a pooled sample from at least six individual rhizosphere soil samples. In same row, different superscript lowercase letters indicated significant differences among different treatments, and same letters indicated no significant difference (p

Targeted Acquisition of Fusarium oxysporum f. sp. niveum Toxin

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