Elsevier

Biological Control

Volume 112, September 2017, Pages 28-33
Biological Control

Oxalic acid produced by Aspergillus niger Y-1 is effective for suppression of bacterial fruit blotch of watermelon seedlings

https://doi.org/10.1016/j.biocontrol.2017.06.001Get rights and content

Highlights

  • Cultural filtrate of Aspergillus niger is effective to suppress BFB.

  • The antibacterial mechanism relies on acidity of the cultural filtrate.

  • Oxalic acid has potential to be exploited as watermelon seed disinfectant.

Abstract

Bacterial fruit blotch (BFB) of watermelon caused by Acidovorax citrulli (Ac) is a seedborne disease. Seed treatment with bacterial disinfectants is considered as an important measure for suppression of Ac infection. This study was performed to detect the antibacterial activity of the cultural filtrate (CF) of the saprophytic fungus Aspergillus niger Y-1 against Ac. The six-day-old CF, citric acid (CA, 4 mmol/L) and oxalic acid (OA, 60 mmol/L) were determined for in vitro antibacterial activity against Ac. CF and OA were determined as seed disinfectants for suppression of seedborne infection by Ac. Results showed that production of CA and OA by A. niger in potato dextrose broth was consistently detected by HPLC. The CF, CA and OA inhibited growth of Ac and their inhibitory effect disappeared when the pH values of the three solutions was adjusted to 7.0. OA was more effective than CA in suppression of Ac. The potting experiment showed that both CF and OA applied on watermelon seeds effectively suppressed BFB incidence on seedlings. The efficacy was comparable to the seed treatment with HCl (1%, v/v). This study suggests that the CF of A. niger and OA can be used as seed disinfectants for elimination of seedborne Ac.

Introduction

Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is a devastating disease on cucurbit crops, including watermelon (Citrullus lanatus) (Schaad et al., 2008, Burdman and Walcott, 2012). The disease was first found in Florida of USA in 1989 (Somodi et al., 1991). Since then, BFB has been spread worldwide and caused great economic losses for cucurbit fruit industries (Burdman and Walcott, 2012). The infected seeds represent the most primary inoculum source of BFB (Rane and Latin, 1992, Hopkins and Thompson, 2002). Seeds even with a low level of A. citrulli containment can result in severe BFB epidemics under the favorable environment (Dutta et al., 2012). Moreover, BFB can cause great economic losses for production of seedlings in commercial watermelon nurseries, where the environmental conditions (high temperature, high humidity) are favorable for infection by A. citrulli (Burdman and Walcott, 2012).

Nowadays, commercial cultivars with resistance to A. citrulli are not available in cucurbit crops (Bahar et al., 2009, Burdman and Walcott, 2012). Therefore, control of BFB mainly depends on use of A. citrulli-free seeds and on treatment of A. citrulli-contaminated seeds either with physical measures such as dry heat or with antibacterial chemicals such as hydrochloric acid (Kubota et al., 2012, Hopkins et al., 2003).

Previous studies showed that selected biological control agents (BCAs) can be used to control BFB on cucurbit crops. The reported BCAs include nonpathogenic strains of A. citrulli and A. avenae (Johnson et al., 2011), antagonistic bacteria such as Bacillus spp. (Santos et al., 2006, Wu et al., 2014), Paenibacillus lentimorbus (Medeiros et al., 2009) and Pseudomonas fluorescens (Fessehaie and Walcott, 2005, Zhou et al., 2009), and antagonistic yeasts such as Pichia anomala (Wang et al., 2009), Rhodotorula aurantiaca and R. glutinis (Conceição et al., 2014, Melo et al., 2015). The proposed mechanisms in biological control of BCAs against BFB include production of antibacterial substances by the BCAs (Fessehaie and Walcott, 2005, Zhou et al., 2009, Wang et al., 2009, Wang et al., 2015), competition of BCAs with A. citrulli through colonization of watermelon seeds and blossoms, or through endophytic growth in watermelon seeds (Fessehaie and Walcott, 2005, Johnson et al., 2011). Treatment of watermelon seeds with non-pathogenic AAC00-1ΔhrcC of A. citrulli (a type III secretion system mutant), A. avenae AAA99-2, P. fluorescens A506, or the cell-free cultural filtrate of P. anomala 0732-1 resulted in effective suppression of BFB incidence on various cucurbit crops (Fessehaie and Walcott, 2005, Wang et al., 2009, Johnson et al., 2011). For example, application of A. avenae AAA99-2 and P. fluorescens A506 to watermelon blossoms could effectively suppress epiphytic growth of A. citrulli, thereby reducing seed infection by A. citrulli (Fessehaie and Walcott, 2005). However, use of selected filamentous fungi or their metabolites to control BFB has not been reported so far.

Aspergillus niger is a filamentous ascomycetous fungus and a common saprophyte widely living in soil and plant debris. It can produce various organic acids, including citric acid, oxalic acid and gluconic acid (Yang et al., 2017). Previous studies showed that A. niger is an effective BCA for control of plant nematodes and oxalic acid was found to be the most toxic compound (Zuckerman et al., 1994, Jang et al., 2016). Whether or not A. niger and oxalic acid can suppress A. citrulli remains unknown. Therefore, a study was conducted to fulfill the following two objectives: (i) to detect the inhibitory effects of cultural filtrate (CF) of A. niger Y-1 and the organic acids (citric acid, oxalic acid) in the CF of A. niger against A. citrulli; and (ii) to determine the efficacy of the CF of A. niger and oxalic acid applied on seeds of watermelon in suppression of bacterial fruit blotch caused by seedborne A. citrulli.

Section snippets

Microbial strains and cultural media

Strain Pslbtw36 of Acidovorax citrulli and strain Y-1 of Aspergillus niger were used in this study. Strain Pslbtw36 was kindly provided by Dr. T. C. Zhao of the Institute of Plant Protection in Chinese Academy of Agricultural Sciences (Beijing, China). Strain Y-1 of A. niger was isolated from a soil sample collected from a field growing with upland cotton (Gossypium hirsutum) in Hubei Province of China (Lu, 2010). The cultural media used in this study included King’s B medium (KB), King’s B

Antibacterial activity of the cultural filtrates of A. niger against A. citrulli

On A. citrulli-KBA cultures (30 °C, 48 h), no clear zones formed around the Oxford cups loaded either with fresh PDB or with the 1-day-old CF of A. niger (Fig. 1). In contrast, formation of clear zones (indication of inhibition against A. citrulli) was consistently observed around the Oxford cups loaded with the 2- to 7-day-old CF of A. niger with the average diameters ranging from 8 to 15 mm. Diameter of the clear zones (CZ) caused by the CF of A. niger was positively correlated with the

Discussion

This study revealed that the CF of A. niger Y-1 could inhibit growth of A. citrulli. This result suggests that the CF of A. niger has antibacterial activity. However, when the pH value of the 6-day-old CF of A. niger was adjusted from 1.6 to 7.0, the antibacterial activity of the CF disappeared. Therefore, the antibacterial activity of the A. niger CF against A. citrulli is probably caused by certain acidic compounds in the CF. A. niger is well recognized as producer of many organic acids,

Acknowledgments

This research was supported by the research and development special fund for Public Welfare Industry (No. 201203066).

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