Potato appear (Keil., et al., 2010; Hansen et al.,

Potato
blight caused by oomycete Phytophthora
infestans is an economic disease that affects potato (Solanum tuberosum) production worldwide and has persistently caused
significant losses (Hassen, et al.,
2016). Late blight has been described as the most destructive disease of potato
which accounts for a significant economic loss per year and is best known as the
cause of the ‘Irish potato famine’ in the middle of the 19th century
where there was complete destruction of potato crops (Ballvora, et al., 2002; Salaman, 1985; Kamoun et al., 2001).  Potato blight negatively impact world agriculture
production, responsible for significant losses in potato production, thus management
of this disease is crucial because potato is the fourth largest food crop and
an important alternative to major cereal crops feeding the world’s population (Brian
et al., 2009; Yao et al., 2015).  

Ballvora,
et al.; Brian et al., 2009 noted that management of this disease is challenged mainly
because of the speed of adaptation of Phytophthora
infestans to various control methods. Thus it is important to develop
methods and techniques which allows early detection of late blight in potato to
avert further destructive losses and assist in proffering effective control
strategies.  This goes a long way in
limiting the spread of the pathogens and reduces severe economic and
sociological losses (Tomlinson et al.,
2009).

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It
is crucial to develop assays for quick detection of the fungus before symptoms
begin to appear (Keil., et al., 2010;
Hansen et al., 2016; Ammour, et al., 2017). It is also important to
note that potentially early detection of the pathogen inoculum before planting
can go a long way in preventing introduction of infected materials into fields
thus effective for management of blight on the field. This as well has been
proven to reduce severe losses due to blight on the farm (Trout et al., 1997; Keil et, al 2010; Judelson, and Tooley, 2000).

Report
shows that there has been a significant yield gap between organic and
conventional production of potato and mainly attributed to lack of adequate measures
to control diseases and pests late blight in particular (Finckh et al., 2006).
This is largely due to deviation from practices such as non-application of synthetic
fertilizers, use of green manure, crop rotation, prohibition of pesticides and
chemicals in organic potato production. Severity of damage by late blight in
conventionally-managed potato farm is lower because of application of synthetic
chemicals and use of various management practices (Struik, 2009, Finckh et al., 2006, Palmer et al., 2013, Eyre et al., 2010).

Several
traditional detection methods which includes physical examination of the tubers
or more recently culturing of plant tissues often are not sufficient for a
robust diagnostic of the fungus exhibiting asymptomatic infections. These
diagnostic methods also fail to effectively detect similarities of symptoms
between the main causative agent and other pathogens (Judelson, and Tooley, 2000).

Several
attempts have been made in developing modern diagnostic methods (DNA-based
diagnostics and DNA sequencing) for rapid detection and diagnosis of Phytophthora and Phytophthora infestans to date, and these have proven effective in management
of plant disease because it helps in early detection of pathogens thus avert
further losses by proffering effective control measures. Prominent amongst
these are use of conventional PCR, Real-time PCR for early detection, (Trout et al., 1997; Judelson, and Tooley,
2000; Martin et al., 2012; Lees et al., 2012; Hussain et al., 2014).

Advances
on existing assays and detection methods have been developed recently. This
include colorimetric LAMP assays for detection of P. infestans a closed tube reaction where naphthol blue was used
for detection (Hansen et al., 2016),
specific assays for P. infestans with
varying detection methods ranging from turbidity, florescence and colorimetry
(Chen et al., 2013; Dong et al., 2015; Goto et al., 2009) and development of onsite detection and real time
isothermal amplification assays (Ammour et
al., 2017).  

A great percentage of
existing diagnostic methods for P.
infestans require thermal cycling and gel electrophoresis but LAMP requires
isothermal conditions and use of small and portable instrument thus afford the
opportunity to rapidly detect P.
infestans within a short time frame and efficiently (Judelson and Tooley,
2000). 

The
use of LAMP for detection of varieties of pathogens has increased greatly in
the past few years largely due to its minimal need for sophisticated equipment,
proven ability to give rapid results, sensitivity and specificity. This DNA
amplification method is on auto cycling strand displacement reaction which
involves use of four to six primers and DNA polymerase (Bacillus stearothermophilus) to facilitate the
reaction under isothermal conditions (Notomi, et al., 2000, Hansen et al., 2016;
Ravindran, et al., 2015).

The
need to develop less sophisticated, inexpensive and fast techniques for
detection of pathogens remains the rationale for Loop-mediated isothermal
amplification (LAMP). This rapidly amplifies target DNA, characterised with
specific and efficient results under isothermal conditions over a range of 60
-65°C and this employs a strand displacement reaction (Notomi et al., 2000).

Objectives

This
study aims to generate a rapid, sensitive method for detection of Phytophthora infestans using
loop-mediated amplification (LAMP) assay and use this to test for variations of
Phytophthora infestans under
conventional and organic management systems.

To
achieve this aim, the following objectives are important:

i.                
Isolation of fungi (Phytophthora infestans) from potato obtained from the field, green
houses and other sources

ii.              
Compare assays specific to Phytophthora infestans using real-time
LAMP

iii.            
Ascertain the sensitivity of the assays
using infected plant materials

iv.            
Test assay specificity using different fungal
species including Phytophthora

v.              
Use the developed assays to ascertain
variations in potato leaves from varying management systems

Proposed Method

Fungal isolates

Fungal isolates (Phytophthora infestans, other species of Phytophthora and different fungi) will be obtained from different sources (Laboratory). Fungal
cultures will be maintained on PEA agar this will be further grown in pea broth
at ambient temperature for 5 – 10 days before being collected for DNA
extraction. Fungal DNA extraction will be done using DNeasy Plant Mini Kit
according to manufacturer’s instructions (Qiagen). Quantification of DNA of
fungal isolates and that of Phytophthora
infestans will be done using a NanoDrop 1000 spectrophotometer (Thermo
Fisher Scientific, Inc. Wilmington, NC). Phytophthora
infestans will be inoculated into potato leaves grown in the green house
and DNA

LAMP Primer Design

Primers
to be used in the LAMP assays are as described by Ammour et al., 2017.

LAMP Reaction

LAMP
reactions will be performed in 25µl reaction mixtures using the isothermal
MasterMix without intercalating dye. 1.6µM Forward and backward internal
primers, 0.2 µM of F3 and B3 primers, 0.8 µM loop forward primer, 0.08µM
assimilating fluorescent loop backward probe strand, 0.12µM assimilating probe
Q-strand and 3 µl of template DNA. Amplification will be carried out for a
period of 30 mins at 65°C while the florescence will be monitored in real-time
using Genie II instrument (OptiGene) (Ammour, et al., 2017).

LAMP Specificity and Sensitivity

To
ascertain the specificity of the LAMP assay, it will be tested on isolates of Phytophthora infestans, other potato
fungal pathogens and those obtained from the field (organic and conventional
potato farm) and a control (nuclease-free water).

To
evaluate the sensitivity of the LAMP assay, 10-fold serial dilutions of pure P.
infestans DNA will be performed and replicated. The reaction will be monitored
by real time-turbidity detection, readings taken, and results will be
interpreted.

Variations in disease incidence

To
ascertain the incidence of P. infestans
under varying management systems, samples will be collected from Nafferton farm
long-trial experiment. Both symptomatic and asymptomatic samples will be used, plant
parts (tissues) will be plated on nutrient agar as described above and fungal
isolates will be obtained from the culture. Fungal DNA extraction will be
performed as described earlier. The developed LAMP assay will be used for
diagnostics of P. infestans on the
collected samples to determine the disease incidence.