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Transformation of Cyclaneusma minus with Green Fluorescent Protein (GFP) to Enable Screening of Fungi for Biocontrol Activity
Institute of Molecular BioSciences, College of Sciences, Massey University, Private Bag 11–222, Palmerston North, 4442, New Zealand
Bio-Protection Research Centre, P.O. Box 84, Lincoln University, Christchurch 7647, New Zealand
Current Address: Scion, New Zealand Forest Research Institute Ltd., Private Bag 3020, Rotorua, 3046, New Zealand.
* Author to whom correspondence should be addressed.
Received: 23 November 2011; in revised form: 10 January 2012 / Accepted: 19 January 2012 / Published: 1 February 2012
Abstract: Cyclaneusma needle-cast has a major impact on the New Zealand forest industry. The causal agent, Cyclaneusma minus, causes most severe damage to 11–20 year-old trees and currently there are no economically viable procedures for control of the disease in New Zealand. Here we present a method for genetic transformation of C. minus using protoplasts generated by incubation with Glucanex™ enzyme. C. minus was transformed with a gene encoding green fluorescent protein (GFP) and expression was stable after successive sub-culturing of the strain in the absence of selective pressure. Expression of the gfp gene allowed us to utilize an in vitro GFP-based screening method to identify strains of Trichoderma with potential for biocontrol of this disease. The strain that showed the most promise as a potential biocontrol candidate exhibited a low level of inhibition by uncharacterized metabolite(s) that C. minus secretes into the medium, and consistently caused a loss of GFP expression from the GFP-labeled C. minus strain. The interaction between C. minus and the biocontrol strain, in the interaction zone where GFP expression was lost, was determined to be fungicidal. The utility of such biocontrol strains is discussed. This study represents the first genetic manipulation of C. minus and will pave the way for further studies of the life cycle and infection biology of this organism.
Keywords: Cyclaneusma; needle-cast; protoplast; transformation; antagonism; biocontrol; GFP
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McDougal, R.; Stewart, A.; Bradshaw, R. Transformation of Cyclaneusma minus with Green Fluorescent Protein (GFP) to Enable Screening of Fungi for Biocontrol Activity. Forests 2012, 3, 83-94.
McDougal R, Stewart A, Bradshaw R. Transformation of Cyclaneusma minus with Green Fluorescent Protein (GFP) to Enable Screening of Fungi for Biocontrol Activity. Forests. 2012; 3(1):83-94.
McDougal, Rebecca; Stewart, Alison; Bradshaw, Rosie. 2012. "Transformation of Cyclaneusma minus with Green Fluorescent Protein (GFP) to Enable Screening of Fungi for Biocontrol Activity." Forests 3, no. 1: 83-94.