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Screening of Alternaria alternata causing Brown Spot Of Potatoes in South Africa

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joel dube

on 16 July 2013

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Transcript of Screening of Alternaria alternata causing Brown Spot Of Potatoes in South Africa

Screening for reduced sensitivity to QoI fungicides in Alternaria alternata isolates from potato in South Africa.
By Joel Prince Dube
Introduction
Get to know me...
Under severe conditions they later coalesce and form large necrotic lesions resulting in premature defoliation
Elongated, superficial brown or black lesions may also form on stems and petioles (Kirk and Wharton, 2012)
occurs over a wide range of climatic conditions
Life Cycle
Problem statement
Causes of Resistance
A Closer Look at the Details
strong evidence that exclusive use of Strobilurin fungicides contributed to the shifting of Alternaria populations from wild type to the mutant resistant type
Field resistance of strobulins has been reported on different pathogens from different countries that had also registered the fungicide
LOL
it'll be in the slideshow for like .000001 seconds
you should keep this here.
QoI resistance was shown to be conferred by point mutations within the CYTB gene

The G143A mutation is the most common mutation discovered to date in QoI-resistant pathogens recovered from field surveys

A point mutation at position 143 of the CYTB gene caused by a base substitution and resulting in an amino acid change from Guanine to Alanine
Research Objectives
Confirmation by PCR
Materials and methods
Supervised by Dr Jacquie Van der Waals
Brown spot of potatoes is now widely recognised to be caused by Alternaria alternata (Fr.) Keissler

It was first reported in South Africa in 2011 (Malrose)

Has a wide host range.

Is a ubiquitous necrotrophic fungus (Stevenson et al, 2001)

It causes small circular brown necrotic spots which start on the abaxial side of leaves
Management: Chemical control with Strobilurins
a broad spectrum
Retention on leaf
Growth enhancement
Phytotoxicity
Mode of action of strobilurins
Quinone outside inhibitor (QoI) fungicides [FRAC] (group 11)
Inhibiting electron transport in the cyt b complex of mitochondria.
Inhibit spore germination, fungal growth, and reproduction
Single site mode of action
Screen A. alternata isolates collected from different potato growing regions of South Africa for sensitivity to Azoxystrobin
investigate possible mechanism of azoxystrobin resistance, if any, in these A. alternata isolates by comparing the cyt b gene from sensitive and resistant isolates
Results
Collection, identification and purification of Isolates
In vitro determination of isolate sensitivity to Strobilurin fungicides
Sequence analysis of the partial cyt b gene from Alternaria isolates
Collection, identification and purification of Isolates
Infected plant material were collected from various fields across South Africa
Isolations: 1% NaOCl, V8, Incubate 25°C
Isolates were identified using PCR with A.alternata specific primers
A technical grade QoI fungicide (AMISTAR®) was dissolved into acetone
Water agar was amended with 0, 1.0, 2.5, 5.0, 10, 25, 50, 75, 85 and 100 µg of azoxystrobin per ml of media
The media was amended with 100µg/ml SHAM to inhibit alternative oxidase pathways
Plates were incubated at 25°C in a completely randomized design under darkness for 16hrs
A 0.1µl aliquot with 1x104 conidia/ml of each isolate was transferred to amended media
Hundred conidia per plate were examined and percentage conidia germinated was recorded
A partial cyt b gene was amplified using primer pair CBF1 and CBR2
Fragments were cloned and transformed to a pGem easy vector and multiplied in E.coli
Resulting fragments were purified and sequenced


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In vitro determination of isolate sensitivity to Strobilurin fungicides
Full transcript