Optimizing blackleg management with fungicides: Fungicide sensitivity and dispersal of
blackleg in canola fields in North Dakota
($53,022 + 14,955 = $67,977)
PI:
Luis del Rio
Introduction
Blackleg of canola, caused by the fungus Leptosphaeria maculans, has been effectively managed
with use of genetic resistance in North Dakota. However, the detection of blackleg strains with
new pathogenicity profiles, like PG3 and PG4 (Bradley et al., 2005), has increased the risk of
occurrence of severe blackleg epidemics tremendously. Further, greenhouse evaluations of more
than 70 commercial canola hybrids indicate all of them are susceptible to some of the new strains
(Marino and del Rio, 2010). Currently, only two compounds with similar mode of action are
registered for control of blackleg (McMullen and Markell, 2010); and while they are effective,
their frequent use and misuse have result in buildup of fungicide resistant-strains in other species
(Wise et al., 2009). Since blackleg reproduces both sexually and asexually, the risk of resistance
to build up in the future will be conditioned to the frequency with which these compounds are
used. Thus, judicious spraying will be required to avoid unnecessary applications. Among the
strategies that could be used to protect fields and still prevent unnecessary exposure to the
chemical is the application of the fungicide in areas that are a higher risk of infection, like the
bordering areas between infected and non-infected fields rather than making blanket applications
in entire fields. To make this a practical alternative, however, epidemiological studies that will
estimate the effective distances to which spores of blackleg can move are necessary. In this
project we propose to conduct such studies.
Current Work
Azoxystrobin and pyraclostrobin are the only compounds registered for control of blackleg in
North Dakota (McMullen and Markell, 2010). Both compounds inhibit mitochondrial respiration
and are classified as quinone outside inhibitors (QoI). Resistance to these compounds has
developed within few years of using them in multiple applications within a single growing
season (Avenot et al., 2008; Wise et al. 2009). While blackleg control with fungicides would
take place very early in the season, these compounds are also registered for control of Sclerotinia
stem rot (SSR), which would take place at flowering time. Use of one of these compounds for
blackleg and the other for SSR control could give growers the false impression they are
“alternating” fungicides, when in reality they would be unintentionally exposing the blackleg
pathogen to a second attack in the same biochemical process.
Sensitivity of blackleg isolates to azoxystrobin or pyraclostrobin is lacking. Numerous reports
have been published on resistance to these compounds (Avenot et al., 2008; Vincelli and Dixon,
2002; Wise et al., 2009) but no work has been done on L. maculans. Azoxystrobin has been
registered for use on blackleg since approximately 2006; however, its use has been rather limited
because blackleg epidemics have been neither generalized nor severe in most canola growing
areas. This would allow us to estimate a sensitivity that would be close to a true baseline of
sensitivity.
Information on the dynamics of blackleg spore dispersal is limited and inconclusive. In
Australia, Aubertot et al. (2004) could not find a spatial gradient of blackleg in plots that were 51
m wide and 30 m long, and concluded that dispersal may be occurring at distances greater than
the ones they used; later, Aubertot et al. (2006) using computer modeling estimated that a 400 m
(1200 ft) separation is desired to protect fields considering a source of inoculum 50 x 50 m in
size and with a potential to produce 1.7 x 109 ascospores m2. In the simulation, Aubertot et al.
observed that spore concentrations would be reduced by almost 99% at that distance, although
some spore would still be able to travel much farther. In Manitoba, Canada, Guo and Fernando
(2005) measured disease gradients from a 25 x 25 m canola residue source and concluded that
incidence and severity declined by 50% within approximately 15 m and 6 m, respectively, from
the source. Further, Guo and Fernando (2005) claimed they could not detect ascospores beyond
25 m from the source and pycnidiospores beyond 45 m from the source. Based on their findings,
they suggested that a 50-100 m distance (150-300 ft) would be enough to protect fields from
incoming inoculum. This estimation seems more applicable to our conditions, although their
results need to be validated.
Objectives
The goals of this project are to characterize the sensitivity of blackleg isolates to fungicides
registered for its control in North Dakota, and to study the dynamics of spore dispersal of
blackleg from sources of inoculum. The project will have duration of three years.
Rationale and Significance
Blackleg is a very important disease that has the potential to decimate susceptible canola crops.
The development and increase in prevalence of blackleg strain capable of infecting most
commercial canola genotypes is reducing the choices available for growers to manage this
disease to the use of fungicides and longer crop rotations. With increased demand for canola
from crushing plants and prospects of higher market prices, it is less likely that growers will
embrace the latter as a part of their management programs. It is likely, however, that some
growers be tempted to make repeated applications of these compounds during the same growing
season, increasing in this way the risk of development of fungicide-resistant strains.
The establishment of an effective fungicide concentration at which 50% of spore germination is
inhibited (EC50) will provide us with a reference value to compare and determine whether
fungicide resistance is building up in the blackleg population. The study of blackleg dispersal
gradients will allow us to determine an effective distance that could be used by growers to
protect their fields from incoming inoculum in case their neighbors have fields where the new
strains are prevailing.
Approach
Objective 1. Determination of an EC50 concentration: Sensitivity to azoxystrobin and
pyraclostrobin will be evaluated by quantifying germination of pycnidiospores suspended in a
water solution amended with fungicide at 0, 0.001, 0.01, 0.1, 1.0, and 10 ppm. A preliminary
study will be conducted to determine the optimum temperature and time of incubation to
measure spore germination. Also, it has been established that some fungi can resort to an
alternative respiration pathway in the presence of respiration inhibitors like azoxystrobin (Ziogas
et al., 1997), and that salicylhydroxamic acid (SHAM) can be used to prevent these fungi from
using an alternative pathway in the presence of azoxystrobin. If SHAM does not affect blackleg
spore’s germination when used alone, we will combine it with different concentrations of
azoxystrobin to obtain a more accurate estimation of the sensitivity of the isolates to the
fungicide. Optimum conditions will then be provided to spores suspended in the fungicide
concentrations and spore germination will be quantified by counting the number of spores that
produced a germ tube at least as long as the length of the spores. Blackleg isolates collected in
previous year will be used for this study, although additional isolates will be collected during the
life of the project.
Expected results
It is expected that information on the relative sensitivity of blackleg isolates will be produced.
We already have numerous blackleg isolates although our earliest isolates were collected in
2004.
Pitfalls and limitations
We do not anticipate many difficulties in achieving this objective. We will make arrangements
with the manufacturers to obtain technical grade samples of both compounds. Most of the work
will be conducted in laboratory under controlled conditions, thus no significant delays are
expected. We will continue collecting blackleg isolates in coming years to
Project timeline
This project has duration of three years. Activities to fulfill the first objective will be conducted
in the first year of the project, although sample collection will continue taking place in the next
two years of the project.
Objective 2. Blackleg dispersal studies: The study of blackleg dispersal will be conducted in two
commercial fields in Cavalier County. Selected fields will be planted to canola in 2011 using a
commercial canola genotype but will have to be neighboring a field that was heavily infected
with blackleg in the 2010 growing season. If at all possible, the fields that will serve as sources
of inoculum will be to the north of the field to be planted to canola in 2011. The canola fields
will be sprayed with azoxystrobin (Quadris) at the recommended commercial doses following a
band pattern running parallel to the source of inoculum. The fungicide will be applied when the
canola are at the cotyledon stage. The band closest to the source of inoculum will be 150 ft long,
and successive bands will be 50 ft shorter than the previous, but all bands will be 90 ft wide. A
total of three bands will make a set. A second set of bands will be established in the same field
some 50 ft away from the end of the longest band of the first set (Figure 1). By applying
fungicides in a pattern we will be able to evaluate disease control in treated areas and compare
that to non-protected areas at different distances from the source.
Four transects running perpendicular to the bands will be established in each set for a total of
eight transects per field. Transects will be located approximately 50 ft apart starting in the middle
of the shortest band. Flags will be placed at 90 ft intervals starting at the border between the two
fields. If the fields are separated by a ditch or road the distance between the edges of both fields
will be measured and the band patterns adjusted to reflect the additional distance. At each station
30 plants will be sampled for disease incidence and severity one week prior to swathing (Figure
1). Four spore samplers will be placed along the fourth transect as shown in figure 1 and
operated continuously from the time canola plants emerge until they reach the four leaf stage.
These samplers will be used to gather information on ascospores gradients.
Figure 1. Schematics of fields illustrating areas to be used to study blackleg dispersal gradients
Expected results
If blackleg epidemics develop we should be able to identify gradients of spore dispersal as well
as gradients of disease incidence and severity along transects.
Pitfalls and limitations
As with any field trial, disease incidence and severity will be influenced by environmental
conditions, amount of inoculum present in the field serving as source, and the genetic
composition of the canola planted in 2011. We have identified growers whose fields were
heavily affected by blackleg in the 2010 season and made arrangements to set these plots in
adjacent fields. We have sampled isolates of blackleg from their fields and confirmed them as
PG3, thus we anticipate any commercial cultivar planted this year will be relatively susceptible
to blackleg.
Project timeline
This project has duration of three years. The first year of this study will 2011, plots will be
established in early May. Spore sampling will start when seedlings emerge and will continue
until plants reach the 4th leaf stage. Disease incidence and severity will be assessed at swathing.
At the end of each year we will analyze the data and calculate gradients if epidemics develop.
Outreach/ Extension activities:
Findings of this project will be shared with growers at field days and winter meetings. Results of
our project will also be shared with the scientific community through presentations at
professional meetings.
Budget narrative
This is a three year project, but funds are being requested for the first year (2011) only.
Salary and fringe benefits:
2011-2012: One year and one month of salaries are requested for one graduate student ($19,440)
at Fargo and one research assistant ($2,714) at Langdon, respectively. Fringe benefits at NDSU
for these personnel are calculated at 2% and 36% of salaries, respectively. Also, one and two
months of salaries are requested for one hourly personnel at Langdon ($1,871) and Fargo
($3,840), respectively. Hourly personnel help in daily research activities. The fringe benefits for
these personnel are calculated at NDSU at 17% of salaries.
2012: 2013: One year and one month of salaries are requested for one graduate student ($20,412)
at Fargo and one research assistant ($2,850) at Langdon, respectively. Fringe benefits at NDSU
for these personnel are calculated at 2% and 36% of salaries, respectively. Also, one and two
months of salaries are requested for one hourly personnel at Langdon ($1,965) and Fargo
($3,840), respectively. Hourly personnel help in daily research activities. The fringe benefits for
these personnel are calculated at NDSU at 17% of salaries.
2013-2014: One year and one month of salaries are requested for one graduate student ($21,433)
at Fargo and one research assistant ($2,992) at Langdon, respectively. Fringe benefits at NDSU
for these personnel are calculated at 2% and 36% of salaries, respectively. Also, one and two
months of salaries are requested for one hourly personnel at Langdon ($2,063) and Fargo
($4,032), respectively. Hourly personnel help in daily research activities. The fringe benefits for
these personnel are calculated at NDSU at 17% of salaries.
Travel:
2011-2012: Travel funds for the PI are requested to defray costs of making six visits to
experimental plots during the growing season, and to attend a professional meeting ($700) where
results of this research will be presented. Also, travel funds in the amount of $2218 are requested
for Langdon personnel to visit the experimental plots twice a week. These funds will allow us to
set the experimental plots, serve the spore samplers, conduct the field sampling, and bring
materials to the lab.
2012-2013: Travel funds to be requested in 2012 will be increased by 5% that of 2011.
2013-2014: Travel funds to be requested in 2013 will be increased by 5% that of 2012.
Materials and supplies:
2011-2012: We are requesting funds in the amount of $16,350 to acquire three volumetric spore
samplers in the first year of the project. The estimated cost of each sampler is $4,850. We have
secured another set of samplers for this first year. These samplers are necessary to obtain
accurate information on spore concentrations at different distances from the sources of inoculum
and to establish associations between that and disease intensity. Also, we are requesting $1800 to
cover cost of laboratory supplies (agar, petri dishes, pipette tips, SHAM) that will be required to
conduct the fungicide sensitivity tests in Fargo and $102 for Langdon to acquire a new battery to
operate one of the spore samplers we already have.
2012-2013: We will request funds to acquire another set of three spore samplers. These samplers
will replace the ones we borrowed from another project in 2011. We included a request for
$1890 to cover laboratory supplies in Fargo and $102 to replace a battery for a spore sampler.
2013-2014: Funds to be requested in 2013 will cover laboratory supplies in Fargo ($1985) and
replacement of a battery to operate a spore sampler ($107).
Publication costs:
2011-2012: $650 is requested to help defray cost of publication of research results from the
project. This includes poster preparation and publication of an article in a refereed journal.
2012-2013: Funds to be requested ($683) will help defray cost of publication of research results
from this project.
2013-2014: Funds to be requested ($717) will help defray cost of publication of research results
from this project.
Other direct costs:
2011-2012: A request for $1,000 (Langdon) is made to cover cost of rental space in two
commercial grower’s fields ($500 for each field).
2012-2013: A request for $1,000 (Langdon) is made to cover cost of rental space in two
commercial grower’s fields ($500 for each field).
2013-2014: A request for $1,000 (Langdon) is made to cover cost of rental space in two
commercial grower’s fields ($500 for each field).
Indirect cost:
NDSU charges 28.205% indirect costs to each project each year. This charge has been authorized
by the funding agency.