Optimising Agrobacterium -mediated transformation of Corynespora cassiicola to deliver DsRed
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ORIGINAL PAPER
Optimising Agrobacterium‑mediated transformation of Corynespora cassiicola to deliver DsRed Murnita Mohmad Mahyudin1 · Gary D. Foster2 · Andy M. Bailey2 Received: 5 October 2019 / Accepted: 27 May 2020 © The Malaysian Rubber Board 2020
Abstract Corynespora cassiicola is a cosmopolitan fungus with a broad host range. One of the hosts often severely impacted is the rubber tree, Hevea brasiliensis on which it causes devastating leaf defoliation. To date, control of the disease is mainly focused on the application of fungicides, but this only provides short-term solution. Functional genomics through transformation allows evaluation of the disease effectors which may eventually lead to more precise control measures to curb the disease. A transformation system for C. cassiicola mediated by Agrobacterium tumefaciens was successfully established with resistance to hygromycin and DsRed as markers. The protocol was optimised with regard to Agrobacterium strains, C. cassiicola isolates, and other parameters. Combination of isolate CLN23 and A. tumefaciens strain LBA4404 carrying pCAMDsRed gave the highest number of transformants. The optimum fungal age, temperature for incubation and co-cultivation period were 7 days old, 28 °C and 4 days, respectively. However, these parameters are highly subjected to interaction between the variables. The transformants carried the DsRed reporter gene, and demonstrated readily apparent red fluorescence, which allowed disease development in planta to be observed. This robust transformation system should pave the way for future genetic manipulations of C. cassiicola. Keywords Agrobacterium-mediated transformation · Corynespora cassiicola · DsRed · Leaf fall disease · Pathogenicity
Introduction Corynespora cassiicola (Berk. & M.A. Curtis) C.T. Wei is a common fungus with world-wide distribution. It is often overlooked and yet it is a plant pathogen with a very broad host range. It has been reported affecting numerous crop species (including tomato, cucumber, basil), but its biggest impact is seen in Hevea brasiliensis, the natural rubber tree. In this host, it is found as a foliar pathogen and causes significant defoliation, commonly known as Corynespora Leaf Fall (CLF) disease. Whilst nursery stocks can be protected by fungicide application, mature trees on a plantation scale pose a more challenging prospect for disease management as epidemics may occur and yields are often severely impacted. * Murnita Mohmad Mahyudin [email protected] 1
Integrated Pest and Disease Management Unit, MRB Experimental Station, Malaysian Rubber Board, 47000 Sungai Buloh, Selangor, Malaysia
School of Biological Sciences, University of Bristol, 24 Tyndall Ave, Bristol BS8 1TQ, UK
2
This is exacerbated by the use of single-clone plantations or mixtures of limited genetic variability, giving a uniform host population, coupled with the ongoing stresses imposed on the plants by repeated tapping to harvest the latex. Understanding the disease progression and the host–pathogen interactions
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