Relative efficacy of strobilurin formulations in the controlof downy mildew of sunflower

J. Sudisha • S. R. Niranjana • S. L. Sukanya •

R. Girijamba • N. Lakshmi Devi • H. Shekar Shetty

Received: 16 July 2009 / Accepted: 12 May 2010 / Published online: 29 May 2010

� Springer-Verlag 2010

Abstract The efficacy of three commercial formulations

of strobilurins, viz., trifloxystrobin, kresoxim-methyl, and

azoxystrobin was evaluated against sunflower downy mil-

dew disease caused by Plasmopara halstedii under labora-

tory, greenhouse, and field conditions. Complete inhibition

of sporangial sporulation, zoospore release, and motility was

observed with 2 lg ml-1 in trifloxystrobin, and 5 lg ml-1

for azoxystrobin and kresoxim-methyl. Seed treatment with

different concentrations of strobilurins enhanced seed ger-

mination and seedling vigor of sunflower to varying degrees

compared to control. Highest seed germination was recorded

at 10 lg ml-1 in kresoxim-methyl, and maximum seedling

vigor was noticed with trifloxystrobin at 30 lg ml-1. The

effect of strobilurins was tested as seed treatment, foliar

application, and seed treatment followed by foliar applica-

tion. Under greenhouse conditions none of the concentra-

tions used, either as seed treatment and foliar application,

were phytotoxic. For the three strobilurins, the seed treat-

ment along with foliar application enhanced the protection of

the plants as compared to only the treatment of seeds. Foliar

spray treatments alone provided an intermediate control of

the disease. Trifloxystrobin showed a better effect than

kresoxim-methyl and azoxystrobin. Disease curative activity

of trifloxystrobin was higher compared to kresoxim-methyl

and azoxystrobin. Tested fungicides when applied on adaxial

leaf surface showed partial translaminar activity, and disease

inhibition was marginal. Loss of trifloxystrobin and azox-

ystrobin activity over time was low, indicating stable rain-

fastness residual activity. A trend in protection against

downy mildew disease similar to greenhouse results was

evident in the field trials. This is the first report of strobilurins

exhibiting high activity against P. halstedii and is a prom-

ising fungicide for controlling sunflower downy mildew

disease by seed treatment and foliar spray.

Keywords Helianthus annuus L. � Plasmopara halstedii �b methoxyacrylic acid group formulations �Seed and foliar treatments � Disease management

Introduction

Sunflower (Helianthus annuus L.), one of the important oil

seed crops, faces the problem of crop loss due to diseases

caused by microorganisms. Downy mildew (Plasmopara

halstedii (Farlow) Berl. & de Toni occurs destructively wher-

ever sunflower is grown. Sunflower downy mildew is reported

in India, and caused severe epidemics in cvs. Morden and EC

68414 in Maharashtra during 1984–1989, and subsequently, in

Karnataka and Andhra Pradesh (Agrawal et al. 1991). It is a soil

and seed-borne pathogen. Mayee and Patil (1986) reported a

field occurrence of the sunflower downy mildew disease in

Marathwada region in Maharashtra state of India, with yield

losses up to 80%. Disease management strategies include use

of resistant hybrids/cultivars, chemical control, and cultural

practices. Sunflower hybrids resistant to downy mildew

are available, but new pathogenic races of the fungus are

being formed in nature, making questionable the use

Communicated by M. Traugott.

J. Sudisha � S. R. Niranjana (&) � S. L. Sukanya �R. Girijamba � H. Shekar Shetty

Downy Mildew Research laboratory, Department of Studies

in Biotechnology, University of Mysore, Manasagangotri,

Mysore, Karnataka 570006, India

e-mail: srn@appbot.uni-mysore.ac.in

N. Lakshmi Devi

Department of Studies in Microbiology, University of Mysore,

Manasagangotri, Mysore, Karnataka 570006, India

123

J Pest Sci (2010) 83:461–470

DOI 10.1007/s10340-010-0316-3

of formerly resistant cultivars in a particular area

(Molinero-Ruiz et al. 2002). Fungicidal seed treatment is a

common practice to control downy mildews and is deemed

economical. The efficacy of metalaxyl against different

oomycetes is a widely reported phenomenon. Metalaxyl seed

treatment (2 g a.i./kg) has been reported to control downy

mildew in sunflower (Patil et al. 1991). Apron 35 SD (6 g a.i./

kg) seed treatment provides maximum control of the sunflower

downy mildew disease (Melero-vara et al. 1982). Fungicides

with definite systemic and long-lasting properties (e.g., met-

alaxyl or related compounds) are of significance in controlling

the disease. Some authors have reported the rapidity with

which resistance to phenylamide fungicides has arisen in nat-

ural populations of oomycetes (Parra and Ristaino 1998).

Resistance to phenylamide fungicides prior to their commer-

cial use has been detected in Pseudoperonospora cubensis,

Phytophthora infestans, and Plasmopara viticola fungicides

(Gisi et al. 2000). The development of metalaxyl-resistant

strains in oomycetes is attributed mainly to genetic uniformity

of the host cultivar and genetic variability in the pathogen

(Parra and Ristaino 2001). However, over-use of the fungicide

has led to the selection of pathogen strains resistant to pesti-

cides, the occurrence of resistant isolates of the pathogen was

first reported under greenhouse (Oros and Viranyi 1984). The

resistance of the oomycete has been found at doses of metal-

axyl lower than 2 g a. i./kg seed in Hungary, USA, and Turkey

(Gulya et al. 1999; Viranyi et al. 1992) and at commercial doses

of 2 g a.i./kg seed in France and Spain (Albourie et al. 1998;

Molinero-Ruiz et al. 2003). Mefenoxam was marketed 10 years

later than metalaxyl, and it contains the biologically active

enantiomer of the racemic fungicide metalaxyl (Shetty 1998).

Both fungicides are used worldwide against sunflower downy

mildew. Resistance to mefenoxam has also been already

reported in natural populations of the oomycetes Peronospora

parasitica and Phytophthora capsici (Parra and Ristaino 1998;

2001; Lamour and Hausbeck 2000). The existence of resis-

tance to mefenoxam in P. halstedii from Spain as well as from

the USA is reported (Molinero-Ruiz et al. 2005; 2008). It is also

suggested that isolates of P. halstedii with resistance to met-

alaxyl may also be resistant to mefenoxam (Molinero-Ruiz

et al. 2005). Different levels of resistance to metalaxyl and

mefenoxam have been observed in P. halstedii with increasing

doses of both fungicides (Molinero-Ruiz et al. 2008).

Pesticides that are adapted to prevent or cure oomycete

plant pathogens either do not exist or are not economically

feasible for marginal farmers (Attard et al. 2007). These

limitations associated with the routinely used strategies

emphasize a search for alternative chemical for sunflower

downy mildew disease management.

Strobilurins, a new class of fungicidal compounds, were

first discovered or isolated from wood-decaying Basidio-

mycete species (Anke et al. 1977). Strobilurins represent bmethoxyacrylic acid group of natural products, which have

become an integral part of disease management programmes

(Bartlett et al. 2002). Strobilurins belong to a new class of

fungicides with a mode of action that is different from that of

the metalaxyl and mefenoxam (Phenyl amide fungicide

(PAF)), iprovalicarb (Carboxylic acid amides) and cyazof-

amid (phenylimidazole chemical class). Mode of action

studies have shown that strobilurins act against mitochon-

drial cytochrome bc1 at the Qo site (Wiggins and Jager

1993) and iprovalicarb does not affect respiration (Stenzel

et al. 1998). Whereas cyazofamid blocks electron transfer in

the mitochondrial cytochrome bc1 complex by binding the

Qi center of the enzyme (Mitani et al. 2001). The most

exciting characteristics about the strobilurins are their

spectrum of activity (Sauter et al. 1999; Herms et al. 2003).

The PAFs are postulated to be site specific, specifically

inhibiting uridine incorporation into RNA polymerase-1

(Sukul and Spiteller 2000), and it was not surprising during

the late 1970s the application of PAFs build up resistance in

susceptible populations (Davidse et al. 1991).

Strobilurins have broad-spectrum activity against Asco-

mycetes, Basidiomycetes, Fungi Imperfecti, and Oomyce-

tes. These fungicides have also been used for the control of

fungal isolates resistant to other fungicides with novel mode

of action and used in low rates (Bartlett et al. 2002). These

strobilurins proved excellent in yield increase and quality of

agricultural produce (Margot et al. 1998; Bartlett et al.

2002; Beck et al. 2002). The recommended applications

include foliar application, seed treatment, and in-furrow

applications. In a short span of time, they have represented

10% of the global fungicidal market (Bartlett et al. 2002)

and led to major changes in disease control programmes by

registering on cereals, turfgrass, grapevines, potatoes, fruit,

nut, and vegetable crops. So far studies with commercially

available strobilurins formulation viz., trifloxystrobin,

kresoxim-methyl, and azoxystrobin to control sunflower

downy mildew have not been attempted.

In this study different experiments were conducted to

evaluate the efficacy of the three strobilurin fungicides on

the pathogen P. halstedii in laboratory. Further, optimiza-

tion of fungicides concentration was carried out based on

their effect on sunflower seed development. The optimized

concentrations were then evaluated both under greenhouse

and field conditions for their effect on downy mildew

disease protection. In addition, the nature and stability of

the test fungicides was also evaluated.

Materials and methods

Test fungicides

Strobilurin fungicides, viz., Trifloxystrobin; benzene acetic

acid; a-(methoxyimino)-2-[[[[1-[3-(trifluoromethyl)phenyl]

462 J Pest Sci (2010) 83:461–470

123

ethylidene] amino]oxy]- methyl]-, methylester, (E,E) (Flint

50 WG) from Bayer, Kresoxim-methyl; methyl (E)-methoxy-

imino-[-(o-tolyloxy)-o-tolyl]acetate) (Stroby 50 WG)

developed by BASF and Azoxystrobin; (E)-2-{2-[6-(2-cy-

anophenoxy) pyrimidin-4-yloxy]phenyl}-3-methoxyacry-

late) (Amistar 250 SC) a product of Syngenta, were used for

this study.

Host and pathogen

Sunflower seeds of the highly susceptible cultivar Morden

were used. The disease response of this cultivar was con-

firmed by screening experiments in the experimental plot

and also under greenhouse conditions. The pathogen

P. halstedii isolated from Morden cultivar and maintained

on the same cultivar under field conditions was used in this

study for all inoculation experiments.

Collection of sporangia and preparation of inoculum

Downy mildew-infected leaves from the Morden cultivar

were collected in the evening, the existing sporangia were

washed in running tap water, and the leaves were cut into

small pieces and placed in humidity chamber prepared by

lining the interiors of petri dishes 50 cm 9 30 cm 9 12 cm

sizes with a wet double layer of blotting paper. These

chambers were kept at 20�C and[95% RH in the dark in an

incubator for 6–7 h. After 7 h, to encourage pathogen

sporulation, the incubator was programmed to 15–18�C the

temperature at which sporangia remain intact and viable for

at least 12 h. Sporangia produced on the leaves were har-

vested into distilled water, the spore load was adjusted to

4 9 104 zoospores ml-1 using a hemocytometer, and used

as a source of inoculum in greenhouse studies (Cohen and

Sackston 1973).

In vitro studies—biological activity

Inhibition of Plasmopara halstedii sporangia

(zoosporangiogenesis)

Downy mildew-infected leaves from Morden cultivar were

collected, the existing sporangia were washed, surface

wetness was removed and 1 cm2 leaf area was smeared

with different dilutions (0.1, 0.5, 1, 2, and 5 lg ml-1) of

azoxystrobin, kresoxim-methyl, and trifloxystrobin for

30 min. Sterile distilled water treatment to infected leaves

(1 cm2) served as control. Leaves were incubated in moist

chambers for 12 h and observed for sporulation. Sporangia

from each treatment were harvested in 1 ml sterile distilled

water and spore load was assessed using a hemocytometer

with four replicates.

Inhibition of Plasmopara halstedii zoospore release and

motility

Sporangial suspension of P. halstedii prepared as per the

procedures of Cohen and Sackston (1973) and adjusted

to 1.5 9 104 sporangia ml-1 using a hemocytometer was

treated with 0.1, 0.5, 1, 2, and 5 lg ml-1 of trifloxystrobin,

kresoxim-methyl, and azoxystrobin in 1:1 ratio (v/v) and

kept for 15 min in dark conditions. Later, the treated spo-

rangial suspension was loaded to a cavity block glass slides

(76 mm 9 25 mm 9 1.25 mm) thick with ground edges

and polished round depression (15 mm diameter and 1 mm

depth) at one side. The experiment consisted of four rep-

licates each. The cavity block slides from each treatment

were observed for zoospore release by counting the empty

and intact sporangia microscopically.

The zoospore suspension of P. halstedii was adjusted

to 4 9 104 zoospores ml-1 using a hemocytometer, then

treated with the above concentrations of the test fungicides

in 1:1 ratio (v/v) and kept for 15 min in dark conditions. To

assess zoospore motility in each treatment they were har-

vested and loaded to cavity block glass slides as explained

earlier. The experiment consisted of four replicates each

(cavity block slides) observed under microscope. Relative

percentage of zoospore motility was calculated based on

numbers of motile zoospores released from the sporangia

in each fungicide treatment and was compared to the

control.

Effect of strobilurins seed treatment on sunflower seed

development

Seeds of ‘Morden’ were treated with different concentra-

tions of trifloxystrobin, kresoxim-methyl, and azoxystrobin

viz., 0.5, 2, 5, 10, and 30 lg ml-1 (400 seeds in 4 ml

solution and shaken until seeds were fully coated). Four

replicates of 100 seeds per treatment were placed equi-

distantly on three layers of moistened blotter disks in the

Perspex plates to evaluate percentage germination (Anon-

ymous 1993) and another set of seeds treated with similar

concentrations were subjected to between paper towel

method. The brown germination paper was soaked in dis-

tilled water, and fifty seeds were placed equidistant apart

on the paper. Another presoaked brown germination paper

was placed onto the first brown germination paper so that

the seeds were held in position. The paper towels were

rolled with polythene wrapping to prevent drying of the

towels, then incubated in an incubation chamber at

25 ± 2�C for 7 days. The experiment consisted of four

replications of 100 seeds (50 seeds in eight towels). Dis-

tilled water treated seeds served as control. After 7 days,

the towels were unrolled and the number of seeds germi-

nated was counted and represented as a percentage.

J Pest Sci (2010) 83:461–470 463

123

Seedling vigor was analyzed after 7 days of incubation, the

length of the root and shoot of each individual seedling was

measured. The vigor index (VI) was calculated using the

formula (Abdul Baki and Anderson 1973).

Vigor index ¼ seed germinationð%Þ � ½Mean root length

þmean shoot length�

The concentrations of the fungicides, which did not have

inhibitory effects on seed germination and vigor index,

were used for further studies.

Greenhouse studies

Effect of seed treatment and foliar spray with strobilurin

fungicides on downy mildew disease incidence upon

inoculation with Plasmopara halstedii

The fungicide treatments consisted of seed dressing with

trifloxystrobin (5 lg ml-1), kresoxim-methyl (5 lg ml-1)

and azoxystrobin (10 lg ml-1), seed dressing at the same

doses followed by foliar spray of trifloxystrobin

(10 lg ml-1), kresoxim-methyl (30 lg ml-1) and azox-

ystrobin (30 lg ml-1) and foliar spray alone. Treated seeds

were sown in earthen pots containing 2:1:1 red soil, sand

(red sandy soil) and manure. Four-day-old seedlings were

whorl inoculated with P. halstedii zoospore suspension

(4 9 104 zoospores ml-1) for three consecutive days once

every morning at around 6 AM. In the whorl inoculation

method, droplets of P. halstedii zoospores were dropped

onto the leaf whorl formed by the emerging seedlings and

allowed to flow down to the base. The challenge-inoculated

plants were maintained under greenhouse conditions

(25 ± 2�C, 95% relative humidity).

Strobilurins foliar spray was applied to inoculated plants

(before disease expression) to near run-off on the 15th day

after seedling emergence. Distilled water seed treatment or

foliar sprayed batches served as controls. Each treatment

consisted of 25 plants in four replications. These were

arranged in a randomized complete block design. Plants

showing downy mildew symptoms such as chlorosis of

leaves, growth of the pathogen and dwarfing were con-

sidered as infected. Disease incidence was recorded when

the plants were 30 days old. Percent downy mildew pro-

tection was calculated using the formula,

Fungicide nature

Curative action

Twenty-one-day old downy mildew-infected (sporulating)

sunflower plants were sprayed with trifloxystrobin (10 lg/

ml-1), kresoxim-methyl (30 lg/ml-1), and azoxystrobin

(30 lg/ml-1) till near run-off and plants were maintained

under greenhouse conditions (25 ± 2�C, 95% relative

humidity). The infected plants sprayed with distilled water

served as control. Each treatment consisted of 25 plants in

four replications. These were arranged in a randomized

complete block design. Observations were made for inhi-

bition of the pathogen in the form of recovery of plants

from disease.

Translaminar activity

The fungicides viz., trifloxystrobin (10 lg ml-1), kresox-

im-methyl (30 lg ml-1) and azoxystrobin (30 lg ml-1)

were applied to 21-day-old downy mildew-infected

(sporulating) plants on adaxial leaf surfaces by surface

smearing, in order to study the translaminar activity. In

addition fungicides were also smeared on the abaxial leaf

surface to study its direct effect on disease and on both

leaf surfaces to study the combined effect. Infected plants

smeared with distilled water served as controls. Each

treatment consisted of 25 plants in four replications and

the experiment was conducted in greenhouse conditions

(25 ± 2�C, 95% relative humidity). The plants were

arranged in a randomized complete block design. The

treated plants were examined for disease symptoms after

5 days of inoculation.

Rainfastness: residual activity

Ten-day old greenhouse plants were sprayed with suspen-

sions of trifloxystrobin (10 lg ml-1), kresoxim-methyl

(30 lg ml-1), and azoxystrobin (30 lg ml-1). Treated

plants were subjected to artificial rain in sleeves by sprin-

kling with tap water for 2 h, dried overnight, and inoculated

after 14 h with P. halstedii (4 9 104 zoospores ml-1).

Distilled water sprinkled plants served as controls. The

% protection ¼ % downy mildew in untreated plant�% downy mildew in strobilurin treated plants

% downy mildew inuntreated plants� 100

464 J Pest Sci (2010) 83:461–470

123

plants were arranged in a randomized complete block

design. The inoculated plants were kept in greenhouse

conditions (25 ± 2�C, 95% relative humidity) and exam-

ined for disease symptoms after 5 days of inoculation.

Field studies

Effect of test fungicides on sunflower downy mildew

disease

Three different field experiments viz., seed dressing, seed

dressing followed by foliar spray, and foliar spray with

the test fungicides, trifloxystrobin, kresoxim-methyl, and

azoxystrobin were conducted in the disease plot in a ran-

domized complete block design with four replicates per

plot, each replicate consisting of four rows of 25 plants.

Each rows with 5 m long and 75 cm apart (between rows)

with 25 cm plant spacing. The disease area had been

infested with oosporic inoculum for over one decade. Field

trials were conducted in the experimental station of

Department of Biotechnology, University of Mysore

(N26�180, E73�300, 817 m altitude, red loam soil) during

monsoon. The concentrations used were the same as in the

greenhouse studies. Foliar application of fungicides was

made on the 10th day after seedling emergence using hand

sprayers. Distilled water treated seed or sprayed plants

served as controls. Downy mildew disease incidence was

recorded 30th day onwards.

Statistical analysis

In vitro studies, biological activity experiments consisted

of four replicates each (hemocytometer and cavity block

slides). In the case of seed development, the experiment

consisted of four replications of 100 seeds, and values were

expressed as mean ± standard error subjected to arcsine

transformation and analysis of variance (ANOVA) using

SPSS Inc. 16.0. Significant effects of treatments were

determined by the magnitude of the F-value (P \ 0.005).

Treatment means were separated by Tukey’s HSD (Hon-

estly Significant Differences) test. Greenhouse experiments

were conducted in 4 replicates of 25 plants each (100

plants), and field experiments were conducted in 4 repli-

cates with each replicate consisting of four rows of 25

plants (400 plants) with randomized complete block design

and were analyzed separately for each experiment and

subjected to arcsine transformation and analysis of vari-

ance (ANOVA) using SPSS Inc. 16.0. Significant effects of

treatments were determined by the magnitude of the F-

value (P \ 0.005). Treatment means were separated by

Tukey’s HSD test.

Results

Biological activity

Inhibition of sporangia

A significant (P \ 0.005) dose-dependent sporulation inhi-

bition was noticed with all the three strobilurin formulations,

at 0.5, 1, 2, and 5 lg ml-1 concentrations (Table 1). Com-

plete inhibition of sporulation was obtained with 2 lg ml-1

of trifloxystrobin, while for azoxystrobin and kresoxim-

methyl it was noticed at the highest concentration

(5 lg ml-1). Further, a significant inhibition of 13000 spo-

rangia cm-2 was noticed in trifloxystrobin at 0.1 lg ml-1.

However, the production of sporangia in treatments with

kresoxim-methyl and azoxystrobin at the lowest dose

(0.1 lg ml-1) was not significantly different from that on the

control.

Inhibition of zoospore release and motility

All the tested concentrations proved to be very effective in

inhibiting zoospore release and arresting zoospore motility

(P \ 0.005) (Table 1). At the lowest concentration of

0.1 lg ml-1, percent of zoospore release of 7, 10, and 26%

and zoospore motility of 61, 52, and 45% were recorded in

treatments of trifloxystrobin, kresoxim-methyl, and azox-

ystrobin, respectively, as compared to that of the control

which recorded 85% zoospore release and 100% zoospore

motility. A dose-dependant inhibition pattern of zoospore

release and motility was observed among treatments.

Trifloxystrobin and kresoxim-methyl showed complete

inhibition of zoospore release and zoospore motility at

2 lg ml-1 concentration and were not significantly dif-

ferent, wherein azoxystrobin treatment showed complete

inhibition of zoospore release and motility at 5 lg ml-1.

Sunflower seed development assessment

Seed treatment with different concentrations of strobilurins

revealed significant (P \ 0.005) enhanced seed germina-

tion and seedling vigor to varying degrees compared to the

control (Table 2). Maximum germination of 82% was

recorded at 10 and 30 lg ml-1 in kresoxim-methyl. In the

case of trifloxystrobin maximum germination of 81% was

noticed at concentrations 2–30 lg ml-1 and was not sig-

nificantly different with kresoxim-methyl (0.5, 2, and

5 lg ml-1) concentrations. While for azoxystrobin seed

treatment, maximum germination of 80% was noticed at

5 lg ml-1, followed by 2 lg ml-1 which recorded 79%

germination. The concentrations 0.5 and 10 lg ml-1 tested

J Pest Sci (2010) 83:461–470 465

123

in this treatment were not significantly different and were

par with the control (78% germination). Further, inhibition

of seed germination was noticed in azoxystrobin treatments

when tested at 30 lg ml-1. A similar trend was noticed for

vigor index in all the tested fungicides. For each of the

fungicide the vigor index of 1599 in control was enhanced

to maximum of 1832 at 30 lg ml-1 of trifloxystrobin, 1758

at 30 lg ml-1 of kresoxim-methyl, and 1688 at 10 lg ml-1

of azoxystrobin.

Greenhouse studies

Effect of seed treatment and foliar spray with strobilurin

fungicides on downy mildew disease incidence

None of the strobilurin treatments produced phytotoxic

symptoms of necrosis, epinasty, hyponasty, wilting, leaf-tip

injury, or vein clearing. Among seed treatments, trifloxyst-

robin was significantly (P \ 0.005) superior when compared

to kresoxim-methyl and azoxystrobin treatments (Fig. 1).

Seed treatment with trifloxystrobin (5 lg ml-1), kresoxim-

methyl (5 lg ml-1), and azoxystrobin (10 lg ml-1) showed

protection of 60, 51, and 37%, respectively, over the control.

Further improvement in protection against disease was

evident when seed treatment with fungicides was followed

by foliar spray. Similar disease protection of 95 and 90% was

evident for trifloxystrobin (10 lg ml-1) and kresoxim-

methyl (30 lg ml-1). Azoxystrobin (30 lg ml-1) offered

72% disease protection, which was highly significant over

the control. In foliar spray treatment alone trifloxystrobin

(10 lg ml-1) revealed significant disease protection of 89%,

followed by kresoxim-methyl (30 lg ml-1) and azoxyst-

robin (30 lg ml-1), which recorded 79 and 68% and were

significantly superior over the control. The disease protec-

tion offered by all the three tested fungicides was highly

significant (P \ 0.005) over the control.

Fungicide nature

Curative activity

When sprayed with trifloxystrobin (10 lg ml-1), kresox-

im-methyl (30 lg ml-1), and azoxystrobin (30 lg ml-1)

till run-off, 21-day old downy mildew-infected sunflower

plants showed varied levels of recovery from the disease

(Fig. 2). Significant (P \ 0.005) disease cure of 92, 80, and

74% was noticed in trifloxystrobin, kresoxim-methyl, and

azoxystrobin treatments, respectively.

Table 1 In vitro effects of three strobilurin fungicides on sporangial production on leaf disk, zoospore release from sporangia, and motility of

Plasmopara halstedii

Treatment Conc. (lg/ml) No. of sporangia (cm2) Percent of sporangia

releasing zoospores

Percent of zoospores

releasing motility

Azoxystrobin 0.1 16000 ± 408 h 26 ± 0.4 h 45 ± 9 g

0.5 12000 ± 478 g 14.2 ± 0.2 fg 31 ± 5e

1 7000 ± 408e 8.7 ± 0.2de 14 ± 8c

2 3000 ± 250c 5 ± 0.4bc 3 ± 0ab

5 0 ± 0a 0 ± 0a 0 ± 0a

Trifloxystrobin 0.1 13000 ± 288 g 6.5 ± 0.4bcd 61 ± 2i

0.5 5000 ± 250d 0.5 ± 0.4a 20 ± 7 cd

1 2000 ± 250bc 0.2 ± 0.4a 11 ± 7bc

2 0 ± 0a 0 ± 0a 0 ± 0a

5 0 ± 0a 0 ± 0a 0 ± 3a

Kresoxim-methyl 0.1 16000 ± 250 h 10.2 ± 0.2e 52 ± 9gh

0.5 10000 ± 288f 3 ± 0.5b 39 ± 6ef

1 5000 ± 288d 0.7 ± 0.2a 14 ± 7c

2 1 ± 0ab 0 ± 0a 0 ± 2a

5 0 ± 0a 0 ± 0a 0 ± 0a

Control Distilled water 17000 ± 629 h 85 ± 0.4i 100 ± 0j

Degrees of freedom 48 48 48

F value 413.0 4418.1 7221.6

P \0.005 \0.005 \0.005

1 Values are means of four independent replications2 Means followed by the same letter(s) within the column are not significantly different according to Tukey’s HSD

466 J Pest Sci (2010) 83:461–470

123

Translaminar activity

Very low downy mildew protection of 14, 15, and 11%

was noticed due to adaxial activity of trifloxystrobin

(10 lg ml-1), kresoxim-methyl (30 lg ml-1), and azoxyst-

robin (30 lg ml-1), respectively, which was highly signifi-

cant over the corresponding controls (Fig. 3). However,

movement of strobilurins from abaxial surfaces was excellent

offering significant disease protection of 80, 76, and 64% in

treatments with trifloxystrobin, kresoxim-methyl, and azox-

ystrobin, respectively. Further improvement in disease pro-

tection of 91, 81, and 72% was noticed with trifloxystrobin,

kresoxim-methyl, and azoxystrobin, respectively, when

applied to both leaf surfaces.

Rainfastness: residual activity

In the rainfastness residual activity tests, significant

(P \ 0.005) disease control of 84 and 66% was evident with

trifloxystrobin (10 lg ml-1) and azoxystrobin (30 lg ml-1),

respectively. Kresoxim-methyl (30 lg ml-1) offered a dis-

ease control of 34% but still remained highly significant

(P \ 0.005) over the control (Fig. 4).

Table 2 Effect of strobilurin

seed treatments on sunflower

seed germination and seedling

vigor

1 Values are means of four

independent replications2 Means followed by the same

letter(s) within the column are

not significantly different

according to Tukey’s HSD

Treatment Conc. (lg/ml) Seed germination (%) ± SE Seedling vigor ± SE

Azoxystrobin 0.5 78 ± 0.4 cd 1600 ± 8hi

2 79 ± 0.2bcd 1619 ± 6 h

5 80 ± 0.2abc 1620 ± 3 h

10 78 ± 0.4 cd 1688 ± 9f

30 77 ± 0.6de 1659 ± 7 g

Trifloxystrobin 0.5 80 ± 0.4abc 1778 ± 5c

2 81 ± 0.2ab 1798 ± 7bc

5 81 ± 0.4ab 1812 ± 5ab

10 81 ± 0.2ab 1826 ± 10a

30 81 ± 0.2ab 1832 ± 11a

Kresoxim-methyl 0.5 81 ± 0.2ab 1697 ± 95f

2 81 ± 0.4ab 1723 ± 10e

5 81 ± 0.2ab 1738 ± 11e

10 82 ± 0.4a 1737 ± 8e

30 82 ± 0.0a 1758 ± 10d

Control Distilled water 78 ± 0.4 cd 1599 ± 7i

Degrees of freedom 48 48

F value 23.17 434.0

P \0.005 \0.005

a

aa

b

b

b

c

c c

dd d0

102030405060708090

100

Seed treatments Seed treatment+Foliar spray

Foliar spray

Treatments

Dis

ease

Pro

tect

ion

(%

)

Trifloxystrobin (SD-5 µg/ml, FS-10 µg/ml)Kresoxim-methyl (SD-5 µg/ml, FS-30 µg/ml)Azoxystrobin (SD-10 µg/ml, FS-30 µg/ml)Control

Fig. 1 Relative performance of strobilurin formulations on sunflower

downy mildew disease under greenhouse conditions

a

bbc

d0

10

20

30

40

50

60

70

80

90

100

Kresoxim-methylTrifloxystrobin(30 µg/ml)

Azoxystrobin (30 µg/ml)

Control

Treatments

Dis

ease

Pro

tect

ion

(%

)

(10 µg/ml)

Fig. 2 Curative activity of strobilurin formulations on sunflower

downy mildew disease under greenhouse conditions

J Pest Sci (2010) 83:461–470 467

123

Field studies

Effect of test fungicides on sunflower downy mildew

disease

Significant (P \ 0.005) downy mildew disease protection

was recorded in all the three experimental fungicides

treatments at varied levels. Seed treatment with triflox-

ystrobin (5 lg ml-1), kresoxim-methyl (5 lg ml-1), and

azoxystrobin (10 lg ml-1) showed a significant protection

of 71, 59, and 50%, respectively, over each of the controls

(Fig. 5). Further improvement in protection against disease

was evident when seed treatment with fungicides was fol-

lowed by foliar spray. Disease protection of 94, 88, and 79%

was evident for trifloxystrobin (10 lg ml-1), kresoxim-

methyl (30 lg ml-1), and azoxystrobin (30 lg ml-1),

respectively, over each of the controls. In foliar spray

treatment alone trifloxystrobin (10 lg ml-1), kresoxim-

methyl (30 lg ml-1), and azoxystrobin (30 lg ml-1)

revealed highly significant disease protection of 91, 83, and

71%, respectively, over each of the controls. All the tested

strobilurins formulations provided highly significant

(P \ 0.005) disease protection over the untreated control.

Discussion

In the current findings trifloxystrobin (2 lg ml-1) exhibited

strong in vitro inhibition of zoospore release and zoospore

motility of P. halstedii followed by kresoxim-methyl and

azoxystrobin both at 5 lg ml-1. These observations are

consistent with those from experiments with Phytophthora

capsici, P. citrophthora, and P. parasitica wherein the

zoospore motility, sporangium formation, and cyst germi-

nation were inhibited at very low concentrations of azox-

ystrobin (Matheron and Porchas 2000). Similar observations

using low concentrations (1–2 lg ml-1) of azoxystrobin

significantly inhibited the host-dependent and -independent

stages of Sclerospora graminicola (Sudisha et al. 2005), and

the sensitivity of Venturia inaequalis to trifloxystrobin was

reported by both in vitro (spore germination) and in vivo

tests on apple seedlings (Farber et al. 2002). Trifloxystrobin

was highly active causing complete inhibition of spore

germination and mycelial growth of Cercospora beticola at

20 lg ml-1 (Karadimos et al. 2005).

The seed development parameters of sunflower were

enhanced in all the tested strobilurin fungicides, and max-

imum seed germination and seedling vigor was observed

with kresoxim-methyl (30 lg ml-1). The strobilurin fun-

gicides, when tested using the non-phytotoxic concen-

trations by seed treatment and foliar spray under the

greenhouse and field studies, were able to inhibit disease

at various levels, and maximum disease inhibition was

noticed by trifloxystrobin compared to kresoxim-methyl

and azoxystrobin.

Seed treatment under artificial inoculation (greenhouse)

and field trials with trifloxystrobin (5 lg ml-1) provided

ccc

b

bb

a

a

a

ddd0

10

20

30

40

50

60

70

80

90

100

Trifloxystrobin Kresoxim-methyl Azoxystrobin

Treatments

Dis

ease

Pro

tect

ion

(%

)Adaxial Abaxial Both Control

(10 µg/ml) (30 µg/ml) (30 µg/ml)

Fig. 3 Translaminar activity of strobilurin formulations on sunflower

downy mildew diseased leaves under greenhouse conditions

a

c

b

d0

10

2030

405060

7080

90100

Trifloxystrobin (10 µg/ml)

Kresoxim-methyl(30 µg/ml)

Azoxystrobin (30 µg/ml)

Control

Treatments

Dis

ease

Pro

tect

ion

(%)

Fig. 4 Rainfastness-residual activity of strobilurin formulations on

sunflower downy mildew disease under greenhouse conditions

a

aa

b

bb

c

cc

d d d0

102030405060708090

100

Seed treatments Seed treatment+Foliar spray Foliar spray

Treatments

Dis

ease

Pro

tect

ion

(%

)

Trifloxystrobin (SD-5 µg/ml, FS-10 µg/ml)Kresoxim-methyl (SD-5 µg/ml, FS-30 µg/ml)Azoxystrobin (SD-10 µg/ml, FS-30 µg/ml)Control

Fig. 5 Relative performance of strobilurin formulations on sunflower

downy mildew disease under field conditions

468 J Pest Sci (2010) 83:461–470

123

superior average disease protection of 65%, followed by

kresoxim-methyl (5 lg ml-1) and azoxystrobin

(10 lg ml-1) which offered 55 and 43% disease protection,

respectively. Trifloxystrobin (10 lg ml-1) as foliar spray

alone recorded higher disease protection (average 90%) than

that of seed treatment, further the combination of both seed

treatment and foliar spray of trifloxystrobin provided highly

significant average disease protection of 94% compared to

individual treatments alone (65% and 90% for seed treatment

and foliar spray, respectively). A similar trend in disease

protection was also observed in kresoxim-methyl and azox-

ystrobin with foliar spray alone and combination treatments.

Trifloxystrobin had stronger curative action (92%) than

kresoxim-methyl (80%) and azoxystrobin (74%). Transla-

minar activity of the tested fungicides was superior mainly

by abaxial modes compared to adaxial means, and this

property of strobilurins has been reported previously

(Hermann et al. 1998; Reuveni 2001). Movement of stro-

bilurins from abaxial surfaces was excellent and offered

significant disease protection in treatments with trifloxyst-

robin, kresoxim-methyl, and azoxystrobin, respectively,

over the control. The foliar uptake of strobilurin fungicides

from both abaxial and adaxial surfaces is rapid due to their

distribution around the application site either by local or

systemic action (Margot et al. 1998). The variation in the

activity of strobilurin fungicides in the above experiments

was due to different biological properties that consequently

confer a wide range of biokinetic behavior both inside the

plants and around its external surfaces (Bartlett et al. 2002).

Strong translaminar activity was recorded by trifloxyst-

robin with high disease protection when applied on abax-

ial-infected leaf surfaces. Similar strong translaminar

activity of trifloxystrobin was observed in sugar beet

infected with Cercospora beticola (Karadimos et al. 2005).

Further, rainfastness residual activity by trifloxystrobin

and azoxystrobin was significant in disease control which

was evident by relatively small loss of both formulations.

Though kresoxim-methyl had good curative activity, it

recorded higher loss of formulation on leaf surfaces resulting

in lesser disease protection. This result clearly indicates the

loss of kresoxim-methyl formulation which was relatively

high compared to trifloxystrobin and azoxystrobin.

In this study all three strobilurin formulations tested

exhibit strong in vitro inhibition of the pathogen, an

increase of sunflower seeds germination and vigor, curative

effect, rainfastness residual activity (except for kresoxim-

methyl), and moderate translaminar activity.

This is the first report of strobilurin fungicides viz.,

trifloxystrobin, kresoxim-methyl, and azoxystrobin exhib-

iting high activity against P. halstedii under laboratory,

greenhouse, and field conditions. These fungicides are very

promising for sunflower downy mildew disease manage-

ment by seed treatment which is feasible and economical,

and they also can be used in foliar treatments. Further

investigations are needed to establish the most efficient

sequential treatments to include strobilurins for sunflower

downy mildew disease management. In conclusion, the

application of strobilurin fungicides as seed treatment and

also by foliar treatments could be a beneficial component

of integrated disease management of sunflower. These

strobilurin fungicides, apart from their action against sun-

flower downy mildew pathogen, are good plant growth

promoters, which is an added advantage for any practical

agricultural system.

Acknowledgments The authors thank Dr. T. S. Thind, Professor,

Department of Plant Pathology, Punjab Agricultural University,

Ludhiana, Punjab, India for supplying the experimental fungicides

and Indian Council of Agricultural Research (ICAR) through All

India Co-ordinated Pearl Millet Improvement Project (AICPMIP),

Government of India for laboratory and field facilities provided at

University of Mysore.

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