Pantoea species as beneficial microorganisms in agriculture

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CHAPTER 2 PA 20, a Semi-Selective Medium for Isolation and Enumeration of Pantoea ananatis

As published in: Journal of Microbiological Methods. 2006. Vol. 64, pages 225-231.

Abstract

A semi-selective medium, PA 20, was developed for the isolation and enumeration of Pantoea ananatis from plant material, specifically from onion seed. The medium has a pH of 8.0 and contains NH4H2PO4, K2HPO4, magnesium sulphate, NaCl, D (+) arabitol, crystal violet, bromothymol blue and thallium nitrate. All P. ananatis strains from a variety of hosts produced characteristic yellow colonies in 6-7 days at 25oC. Plating efficiencies on PA 20 in comparison to nutrient agar ranged from 92 to 112%. Recovery from naturally infested and artificially contaminated onion seed was high, with an almost total reduction of saprophytes.
Keywords: PA 20 medium; Pantoea ananatis, Seed; Selective isolation

Introduction

Pantoea ananatis is a pathogen causing diseases in a number of economically important plants including onion (Gitaitis and Gay, 1997; Schwartz and Otto, 2000), Eucalyptus (Coutinho et al., 2002), corn (Paccola-Meirelles et al., 2001), melons (Bruton et al., 1986; Wells et al., 1987), sudangrass (Azad et al., 2000), rice (Azegami et al., 1983) and pineapple (Serrano, 1928).ananatis is both seed-borne and seed-transmitted in sudangrass (Azad et al., 2000), rice (Tabei et al., 1988) and onions (Walcott et al., 2002). The disease of onion, caused by ananatis, was named center rot. Onion seed associated with the first outbreak of center rot in Georgia, USA (Gitaitis and Gay, 1997), was produced in South Africa and it was suspected that the bacterium was introduced into that country on infected seed lots (Walcott et al., 2002). In South Africa, efforts to control the quality of commercially produced onion seed mainly focus on the detection of fungal pathogens, and little is known about potential bacterial pathogens associated with these seeds. Nutrient Agar (NA) and yeast extract dextrose calcium carbonate (YDC) (Wilson et al., 1967) are the common growth media used to isolate P. ananatis from plant material and seed (Azad et al., 2000; Coutinho et al., 2002; Walcott et al., 2002). These media, however, are non-selective and many other organisms present as saprophytes or endophytes in plant material and in seed may hamper the detection of the target pathogen. In this paper, we describe a semi-selective medium, PA 20, which suppresses growth of many saprophytic microorganisms and serves as a suitable medium for growth and enumeration of P. ananatis. The medium was specifically developed to detect this pathogen on onion seed.

Materials and Methods

Bacterial strains

Bacterial strains used in this study are listed in Table 1. Stock cultures of all isolates were maintained in milk-glycerol liquid medium (10% skim milk, 15% glycerol in distilled water) at –70oC. Stock strains were transferred onto NA (Difco) plates and incubated at 25oC to recover growing cultures. Cultures were routinely checked for purity and colony characteristics.

PA 20 development

Criteria for the semi-selective medium were: differentiation between P. ananatis and other bacteria by colony morphology and inhibition of fungal and bacterial contaminants commonly found on onion seed, without hampering the growth of P. ananatis.
Substrate utilisation profiles using the BIOLOG (BIOLOG, Inc., Hayward, CA) plates designed for gram negative bacteria revealed that P. ananatis strains utilised D (+) arabitol, while the majority of other bacteria listed in Table 1 did not (data not shown).
A variety of media, based on the utilisation of D (+) arabitol and modification of Medium C of Dye (Dye, 1968) were prepared. Different concentrations of NaCl: 0.5; 1.0; 2.0; 3.0 and 4.0%, and a range of pH from 7.0 until 10.0 were evaluated. The pH indicator bromothymol blue was added to monitor pH changes during bacterial growth. Crystal violet and thallium nitrate were added (Ishimaru and Klos, 1984) to suppress other bacteria and fungi (Norris et al., 1976; Srivastava et al., 1976). A loopful of bacterial suspensions of the strains listed in Table 1 was streaked on the media in triplicate and observed daily for bacterial growth.
The medium that satisfied the criteria specified above was named PA 20 and had the following composition per litre: NaCl, 20 g; K2HPO4, 1 g; NH4H2PO4, 1 g; MgSO4 x 7H2O, 0.2 g; bromothymol blue, 1 ml of 1.6% aq. solution; crystal violet, 2 ml of 0.075% aq. solution; agar, 15 g. The pH was adjusted to 8.0 with 1.0 N NaOH. After autoclaving and cooling to 50o C, 3 g of D (+) arabitol dissolved in 5 ml water and 2 ml of 1% aq solution of thallium nitrate were added. Both solutions were filter sterilised. Plates were stored at room temperature for 24 hours before use.

Colony morphology and plating efficiency on PA 20

Sixteen P. ananatis strains were selected to determine colony morphology and plating efficiency on PA 20 (Table 2). The strains were grown on NA for 24-48 hours at 25oC. Colonies were suspended in 9 ml quarter-strength, sterile Ringer (Oxoid) buffer (RB) to an absorbance density of 0.1 at 640 nm (UV-160A Shimazu spectrophotometer). Ten-fold serial dilutions were prepared in half-strength nutrient broth (Difco), and 0.1 ml of selected dilutions was spread-plated in triplicate on PA 20 and a non-selective NA (contained per litre: beef extract, 3.0 g; peptone, 5.0 g; agar, 15.0 g; pH 6.8 ± 0.2 at 25oC). Plates were incubated at 25oC. Colonies were assessed and counted after 7 days. Plating efficiencies were determined by dividing the number of colonies growing on PA 20 by the number of colonies on NA, and multiplying the quotient by 100. The experiment was replicated twice.

PA 20 selectivity assessment

Selectivity was evaluated by assaying pathogen-free onion seed artificially infested with P. ananatis strain Pans 2002-2. One seed lot contained a large number of saprophytic bacteria (5.4 x 106 CFU/gram) while the second seed lot had a low population of saprophytes (2.2 x 103 CFU/gram). For each seed lot two sub-samples, 5 g each, were placed in separate sterile mortars and crashed with pestles. Crashed seeds were transferred to separate sterile Erlenmeyer’s flasks containing 100 ml of RB. One of the sub-samples of each seed-lot was spiked with a suspension of P. ananatis to produce an estimated population of 103 CFU/ml. Flasks were incubated for 30 min at room temperature on a rotary shaker. Ten-fold serial dilutions were made in half-strength nutrient broth, and 0.1 ml of each dilution (spiked and non-spiked) was spread on the surface of PA 20 and NA. Colonies were counted after 7 days incubation at 25oC. Suspected P. ananatis (selection based on colony morphology), were purified and their identities confirmed by: oxidase test, indole production and Hugh-Leifson oxidation/fermentation (Walcott et al., 2002). Tests were replicated three times.

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Isolations from naturally infested seed

Five onion seedlots obtained from seed companies and farmers were tested for the presence of P. ananatis. Seed extracts were prepared as described above. For each sample, ten-fold dilution series were made in half-strength nutrient broth, and 0.1 ml of each dilution and direct seed extract was plated in triplicate on NA and PA 20. Plates were incubated at 25oC and examined daily for the presence of characteristic P. ananatis colonies. Three suspected P. ananatis isolates were selected from each PA 20 plate (selection based on colony morphology) for further identification.

Identification of P. ananatis from naturally infested seed

Isolates were purified and their identities confirmed by pigment production on NA, motility, cell morphology, Gram stain, oxidase, catalase, indole production, Hugh-Leifson oxidation/fermentation, gas production from glucose and hydrogen sulphide from cysteine
(Mergaert et al., 1993). The tests were done according to methods described by Fahy and Hayward, 1983.
Pathogenicity was determined on onion (Allium cepa, cv. Granex 33) in greenhouse assays by using a stub inoculation test adapted from Fenwick and Guthrie, 1969. A sterile needle was dipped into the bacterial colony on NA (24-48 hours growth) and then the needle was inserted under the epidermis of a leaf. At least two leaves were inoculated per isolate. Inoculated plants were incubated in a greenhouse with 27oC/23oC day/night temperatures and observed daily for the development of symptoms.

Results and discussion

PA 20 development

D (+) arabitol as a single carbon source, the pH, NaCl concentration and presence of thallium nitrate all contributed to the PA 20 selectivity. P. ananatis grew on PA media at pH 7.0; 8.0 and 9.0, but some saprophytes grew as well, sometimes producing large, slimy colonies. The addition of thallium nitrate inhibited saprophytic bacteria at pH 9.0 but not at pH 7.0. However, the growth of P. ananatis on PA medium at pH 9.0 containing thallium nitrate was also hampered. Only at pH 8.0 with thallium nitrate did P. ananatis grow well and saprophytes listed in Table 1 were inhibited. The concentration of NaCl influenced the colony size and the time of growth to achieve a colony large enough for recognition and enumeration. When the concentration of NaCl was lower or higher than 2% colonies of P. ananatis were either too small for evaluation (<2%) or took 10 or more days to appear on the media (>2%). Thus the medium, named PA 20, containing thallium nitrate, 2% NaCl and a pH 8.0 was selected for further evaluation.

Colony morphology on PA 20 and plating efficiency

All strains of P. ananatis grew on PA 20 medium and colonies were visible after incubation for 6 to 7 days (Table 1). Colonies were yellow, 3-4 mm in diameter; shiny, drop-shaped with small, granular, darker yellow inclusions within. P. ananatis decreased the pH of the medium (degradation of D (+) arabitol to acid) and produced a diagnostic yellow zone around individual colonies (Fig. 1).
Pseudomonas syringae, Pantoea agglomerans, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas campestris and saprophytes isolated from onion seed did not grow on PA 20 medium. Pantoea stewartii subsp. indologenes produced small, 1-2 mm in diameter, yellow colonies with a dark green centre (Table 1).
Plating efficiencies of the 16 P. ananatis strains ranged from 92% to 112% with a mean of 99.5% on PA 20 medium compared to NA. Results are presented in Table 2

Summary 
Acknowledgements 
Contents 
Preface 
Chapter1   The genus Pantoea in plant pathology 
Introduction
The genus Pantoea
Pantoea species as pathogens
Pantoea species as beneficial microorganisms in agriculture
Isolation of Pantoea species from plants
Determination of pathogenicity
Phenotypic identification of Pantoea species using commercial systems
Detection of plant pathogenic bacteria using the polymerase chain reaction (PCR)
DNA fingerprinting-based methods used for identification and taxonomy of bacteria
16S rRNA gene sequence analysis
Multi-locus sequence analysis (MLSA)
Integration of various diagnostic methods for a polyphasic identification
Conclusions
Literature cited
Chapter 2 PA 20, a semi-selective medium for isolation and enumeration of Pantoea ananatis
Abstract
Introduction
Materials and methods
Results and discussion
Acknowledgements
References
Tables
Figures
Chapter 3 Isolation and identification of Pantoea ananatis from onion seed in South Africa
Summary
Introduction
Materials and methods
Results
Discussion
Acknowledgements
References
Tables
Figures 9
Chapter 4 Isolation and identification of the causal agent of brown stalk rot, a new disease of maize in South Africa
Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgements
Literature cited
Tables
Figures
Chapter 5 Polyphasic characterisation of Pantoea strains from onion and maize and the description of Pantoea allii sp. nov.
Abstract
Introduction
Materials and methods
Results
Discussion
Description of Pantoea allii sp. nov.
Acknowledgements
Literature cited
Tables
Figures
Appendix A
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