chapter 2 effects of repeated treatments of …

CHAPTER 2

EFFECTS OF REPEATED TREATMENTS OFDEMETHYLATION-INHIBITOR (DMI)

FUNGICIDES ON DMI-SENSITIVE POPULATIONSOF RUTSTROEMIA FLOCCOSUM

41

LITERATURE REVIEW

Thousands of compounds continue to be isolated,

synthesized, and screened in hopes of finding

antimicrobial properties. Most of the compounds shown to

be successful in clinical trials were active on bacteria

and not fungi (21). The first uses of antifungal

compounds were restricted to human medicine, followed by

veterinary applications, before being expanded possibly

for the treatment of fungal diseases of plants (44). The

development of compounds that interfere with sterol

function and metabolism within fungal pathogens have

become increasingly prevalent in plant-, animal-, and

human-disease management (11).

The Ergosterol-Biosynthesis Pathway and Sterol-

Biosynthesis Inhibitors

Sterols are important in the maintenance of cell-

membrane integrity, synthesis of bile acids, and the

production of steroid hormones (44). Campesterol,

sitosterol, and stigmasterol are the main sterols found In

plants (10)

mammals (69).

Cholesterol is the predominant sterol in

Ergosterol is the characteristic sterol in

the ascomycetes, basidiomycetes, and deuteromycetes (95),

which are the three classes of the higher fungi (1).

42

Koller classified sterol-interfering antifungal

agents into four groups (44). The first of these was the

polyene microlides, which began with the description of

nystatin in 1950 (34). Amphotericin B followed in 1956

(29) and became the most useful of the polyene microlides

(21) of which more than 200 compounds were described by

1981 (68). They have been successfully used in the

treatment of candidiasis (23,42). Their mode of action lS

to bind preferentially to ergosterol molecules that

reinforce the cell membrane resulting in a loss of the

membrane's structural integrity with subsequent leakage of

critical lons from within the cell (41,42). No macrolide

products have been developed for use on plants (44).

The other three groups are collectively known as

"sterol biosynthesis inhibitor" (SBl) fungicides

(11,44,71,76), "sterol inhibitor" (Sl) fungicides (64,65)

and "ergosterol biosynthesis inhibitor" (EBl) fungicides

(20,72). All three terms are synonymous. Their mode of

action is to interfere with ergosterol biosynthesis,

thereby affecting cell-membrane maintenance and formation

(73)

The ergosterol biosynthesis pathway lS highly

conserved (44) and can be segmented into three parts. The

first is the formation of squalene; this is common to all

43

sterol production in animals, plants, and fungi (12). The

synthesis of squalene begins with acetyl-CoA and involves

ten enzymatic steps with farnesyl diphosphate being the

immediate precursor to squalene (44).

The second part contains the conversion of squalene

into lanosterol (57). An essential step within this

segment is the formation of squalene epoxide (66). The

newest class of SBI fungicides, the allylamines, inhibits

squalene epoxidase, thus leading to an accumulation of

squalene and a subsequent deficiency of lanesterol (67)

The allylamines are still restricted to the

chemotherapeutic treatment of human mycoses; no uses on

plant pathogenic fungi have been reported (44).

The third segment of the ergosterol biosynthesis

pathway is catalyzed by membrane-bound enzymes and

involves the conversion of lanosterol into ergosterol

(59). Action sites of the other two classes of SBI

fungicides, the azole fungicides and the morpholine

fungicides, occur within this final segment. The azoles

were introduced in the late 1960s and were routinely

screened for potential as pesticides as well as drugs

(44) . The azoles are structurally diverse (97) but share

a common site of action.

44

Specifically, they interfere with demethylation at

the C-14 position of lanosterol by outcompeting 24-

methylene dihydrolanosterol to bind with its respective

enzyme, the 14a-sterol demethylase. This results in an

accumulation of lanesterol, and ergosterol is not

subsequently synthesized (13,45). Accordingly, this class

was called the sterol C-14-demethylation inhibitor

fungicides, or SDIs, by Buchenauer (14) and Lyr (53) in

1987. Scheinpflug and Kuck referred to them more

concisely in the same monograph as the demethylation

inhibitor (DMI) fungicides (47), and this name became

commonly used (27,28,31,71,75,89,91).

The morpholines were first utilized in agriculture

before later being developed as antimycotic

pharmaceuticals (44). Its first member, dodemorph, was

introduced in 1965 as the first plant-protective SBI (63),

although its specific mode of action was not understood at

the time (97). Morpholines have dual action sites that

are both within the lanosterol to ergosterol segment of

the ergosterol biosynthesis pathway (40). Both sites

occur after the C-14 demethylation (58) and involve

enzymatic mechanics that are very similar to each other

(44). Specifically, they block 614 double-bond reduction

45

and ~8-~7-isomerization. These actions have the lethal

effect of inhibiting mycelial growth as chitin becomes

irregularly distributed in the cell (58).

The sterol-biosynthesis pathway has been a productive

metabolic area in terms of research, development, and

production of phytofungicides. The SBIs constitute the

largest and most important group of these (24). The most

commercially viable group has been the DMIs (35). It is

also the largest. All products in use today were

described as early as 1992 when Koller listed 40 DMI

fungicides and antimycotic drugs with 23 of them then used

in agriculture (44). The relative ease of patentability

can be attributed somewhat to the diverse-structural

differences among molecules that can inhibit C-14

demethylation. The chemistry of the DMls would have been

broadened much earlier had biochemists recognized this

sooner (13).

DMI Fungicides - Product Development

The DMls are used to manage a broad spectrum of plant

diseases. Schulz and Scheinpflug (74) iterated several

advantages:

(a) They can be used in a prophylactic, curative, or

eradicative manner.

46

(b) They can be applied as sprays, granulars, or

seed treatments.

(c) They control not only individual pathogens but

often whole disease complexes.

(d) They cause phytotoxicity less frequently because

of their systemic nature and specific mechanism

of action.

(e) They make possible low application rates because

of their high fungicidal potency.

The DMI-fungicide class can be further divided into sub-

groups based on structural differences that may account

for their selectivity and variation among themselves.

These groups are the piperazines, imidazoles, pyridines,

triazoles, and pyrimidines.

The first DMI to be introduced was the piperazine

triforine launched in 1969 by Boehringer Sohn Company

(74) . It controls powdery mildew and rusts and is the

sole piperazine used in plant protection (72). The first

imidazole, imizalil, was presented by Janssen in 1972 for

use as a seed treatment to inhibit soil-borne pathogens of

cereals (72,74). The only commercially available pyridine

is buthiobate launched as Denmert by Sumitomo in 1975

(72). Kato reported it having in vitro effectiveness on

several ascomycetes and deuteromycetes (39), but in

47

Its current registrant is Bayer

practice it has been used primarily on powdery mildew In

Japan (72).

In 1973, Grewe and Buchel (33) reported the

effectiveness of fluotrimazole on powdery mildew. Bayer

subsequently introduced it as the first commercially

available triazole fungicide (72,74). Steffens and

wieneke later documented its systemic activity (79).

However, no record could be found of it having been

registered for use in the U. S. (92,93,94).

Triadimefon, another Bayer triazole, was introduced

in the same year and was the first to have broad-spectrum

activity on a number of ascomycetes, basidiomycetes, and

deuteromycetes (72,74). It received U. S. Environmental

Protection Agency (EPA) registration as Bayleton in 1980

(94). Triadimefon set new standards for chemical control

of plant diseases (97) and became the first DMI labeled

for use on turf (38). Registration of the product was

passed on from Mobay to Miles and to Bayer Crop

Protection, which were all subsidiaries of the Bayer

Corporation (9)

CropScience.

The pyrimidines were developed from the pyridines as

plant-protection fungicides (97). The Eli Lily

Corporation developed the only commercial products,

48

triamirol and fenarimol (44). The latter has been the

most successful having been launched into the crops market

in 1975 as Rubigan EC by its agricultural subsidiary at

the time, the Elanco Products Company (3). Rubigan AS was

labeled for turf in 1983 (61). The product was passed

onto DowElanco in 1989 and Dow AgriSciences in 1997 before

being sold to the Gowan Company of Yuma, Arizona, in 2001

(8) .

Rubigan has proven to be an effective fungicide on

many turf diseases. However, it is unique in that 75% of

its sales In recent years have been as a niche-market,

preemergence herbicide (S. Farrington, personal

communication) that can suppress the germination of annual

bluegrass (poa annua L.) in bermudagrass (Cynadon dactylon

[L.] Pers.) golf-course greens and tees overseeded with

perennial ryegrass (Lolium perenne L.), rough bluegrass

(Poa trivialis L.), or creeping bentgrass (Agrostis

stolonifera L.) (88). Current registrant Dow

Agriscience's Supplemental Label D06-027-001 issued by the

EPA stipulates two applications spaced 14 days apart at

least 14-days prior to overseeding with perennial

ryegrass. Rough bluegrass or creeping bentgrass should

receive three applications at 14-day-spray intervals (5)

49

The total dosage of active ingredient (AI) for both

programs is 370 g 100 m-2.

Menn and Beard achieved excellent control with

similar timings using maximum total dosages of only 90 g

AI 100 m-2 with higher rates causing discoloration of

Tifgreen and Tifdwarf bermudagrass (Cynodon dactylon (L.)

Pers. x C. transvaalensis Burt-Davy (54,55,56). No other

fungicides were tested in these studies and no dollar spot

was evident (W. Menn, Personal Communication). The lack

of dollar-spot incidence is understandable since the

maximum-application rate of fenarimol for dollar spot is

less than 50 g AI 100 m-2 every 14 days (7). No reports of

any testing with other DMls have been found. The

uniqueness of fenarimol for this use may be because it lS

the only pyrimidine labeled for turf.

Other triazoles besides triadimefon have been shown

to be effective turf fungicides. Propiconazole was

originally discovered and patented by Jansen

Pharmaceuticals (72,74). Ciba Geigy then acquired the

compound's agricultural-use rights and introduced it in

1979 as Tilt. The company acquired EPA registration

numbers for several more formulations directed toward

different market segme~ts. The product labeled for turf

and ornamentals was released in 1987 and trade-named

50

Banner (4). Ciba-Geigy became known as Ciba in 1992

before it merged with Sandoz in 1996 to form Novartis,

which in turn joined with AstraZeneca to form Syngenta,

which exists today (9).

Cyproconazole was registered as Sentinel 1994 by

Sandoz Crop Protection, but it was sold to Bayer to meet

antitrust requirements set forth by the European

Commission that allowed the formation of Syngenta (6).

The most recent triazole turf-fungicide is trade-named

Eagle. It contains myclobutanil and was registered ln

1997 by Rohm and Haas, which was acquired by Dow

AgriSciences in 2001. Triton with AI triticonazole was

expected to be registered by Bayer for turf use in 2004

(96) . However, it and tetrabutrazole have been delayed

indefinitely due to recent EPA concerns with the

environmental fate of DMI metabolites (R. Hanrahan,

personal communication). No other DMIs are being

developed for use on turf by Syngenta (J. DiPaola,

personal communication), Bayer (R. Hanrahan, personal

communication), Dow (F. Pretzer, personal communication),

or Gowan (S. Farrington, personal communication), at this

time.

Current products labeled for use on U. S. golf

courses are shown in Table 2.1. They may have identical

51

or different trade names when sold elsewhere depending

upon the product and the country. The U. 8. patents for

triadimefon, fenarimol, and propiconazole have expired

resulting in similar products being offered by more than

one company.

DMI-Fungicide Formulations and Efficacies

The DMI fungicides are a class of some of the most

systemically fungistatic compounds available for use on

plant diseases (74). Demethylation inhibitors trans locate

primarily in the apoplast but some symplastic transport

does occur (72). Formulations containing DMIs for spray

applications in diluted forms include wettable powders,

water-dispersible granules, dry flowables, aqueous

suspensions, microemulsions, gels, and emulsifiable

concentrates. Dry applications can be made as granulars

or combined with fertilizers. The DMIs are typically

applied at lower quantities of AI per unit area when

compared to the benzimidazoles and dicarboximides.

The DMIs are single-site (88) fungicides under

multigenic control (85,87). This action site is within

the sterol biosynthesis pathway, which is critical to a

higher fungi metabolism. Ascomycetes, basidiomycetes, and

deuteromycetes produce ergosterol and oomycetes do not,

52

which explains their respective sensitivities and

tolerances to DMIs (43).

DMI Use on Dollar Spot

Those DMIs labeled for use on turf have been

effective in suppressing Rutstroemia floccosum, the causal

pathogen of dollar spot. Most product labels recommend

application intervals of up to 28 days. Golf-course

superintendents in the Upper Midwest reported reduced

control of dollar spot in the late 1990s when using DMIs

(38,89). Shortened lengths of suppression were observed

and could only be restored by increasing application

rates. This was consistent with DMI resistance shown with

other plant diseases (26,46). Golembiewski et al.

documented R. floccosum cross-resistance to the DMIs in

1995 (31).

DMI-SENSITIVE R. FLOCCOSUM STUDY

Combining or alternating different classes of

fungicides has long been touted as a means of preventing

or retarding the shift toward a fungal population

dominated by those resistant to at least one of the

classes having been applied (77,78). Turfgrass

pathologists have often recommendeded tank-mixing or

53

alternating systemic- and contact-type fungicides

(16,22,90). However, Vargas has contended that combining

or alternating different SS-fungicide classes results in

multiresistance to those classes; therefore, it is not a

viable strategy for delaying R. floccosum resistance

(84,86,87,88) .

The objective of the experiment presented here was to

determine if a DMI-sensitive population of R. floccosum

could be shifted to a predominantly DMI-resistant

population by the directional-selection pressure of

repeated applications of a DMI fungicide. Another goal

was to determine any differences that occurred among

applications made solely with a DMI, a DMI combined with a

fungicide of a different class, or a DMI alternated with

such. A subsequent objective was to determine the number

of applications needed to effect any such shifts.

Agronomic Practices and Site Assessment

The study was conducted at the Hancock Turf Research

Center at Michigan State University (MSU) in East Lansing,

Michigan. The experimental area was primarily annual

bluegrass (poa annua L.) with some creeping bentgrass.

The soil was a silty clay loam. Supplemental irrigation

to prevent visual wilt was applied in early evening except

54

following significant rainfall events after which the soil

was allowed to dry down. The turf received nitrogen

applications totaling 19 to 29 kg 100 m-2 annually via five

to eight fertilizer applications. Other plant-available

nutrients were adequate according to soil tests conducted

by the MSU Soil and Plant Nutrient lab.

The turf was mowed at a 13-mm height with clippings

collected and removed from the site. The mowlng frequency

was two to three times weekly during the growing season,

and the direction was parallel to the longitudinal axis of

each plot. The removal of any clippings that remained was

attempted using a lawn sweeper. The plots were arranged

ln a randomized-complete-block design with four blocks.

Each plot measured 2.1 m by 8.4 m or 17.6 m2 (See Figure

2.1) .

Dollar-spot incidence increased in late July 1994 until

the entire plot area was severely diseased. A 25-mm-

diameter soil-probe was used to collect plugs that

included diseased tissue from the middle of each plot.

This area measured 1.2 m by 3 m (3.6 m2). The end-to-end

and side-to-side distances between the sampling areas in

adjacent plots were 4.2 m and 5.8 m respectively. Ten or

more sub samples were taken from each plot. The samples

55

were then stored In a freezer until they could be analyzed

in the lab.

Samples were removed from the freezer as needed and

allowed to thaw at room temperature. Three diseased

leaves from each sample were then placed on plastic Petri

plates containing autoclaved potato-dextrose agar (PDA)

(Difco Laboratories, Detroit MI) that had been cooled to

50 C before being amended with 10 pg ml-1 penicillin and pg

ml-1 streptomycin. Mycelia were allowed to grow for one

week at room temperature (21 C). The pure culture was re-

isolated on PDA and grown for three days.

A 10,000-ppm stock-solution of concentrated fungicide

was prepared by suspending 1 g of the DMI Bayleton 25

(0.25 g triadimefon) in 25 ml of water that had been

purified via reverse osmosis and then autoclaved. One I

of PDA was autoclaved and allowed to cool to 50 C before

0.2 ml of thoroughly-agitated stock-solution was slowly

stirred into the media. The resulting concentration of

triadimefon was 2 pg ml-1. Plates were filled with 15 ml

each of this amended-PDA media.

Seven-mm-diameter plugs of each isolate were placed

on two different 88-mm-diameter plates with one containing

PDA and the other having the triadimefon-amended PDA.

Each plate contained a single plug. The cultures were

56

allowed to grow at room temperature until the mycelia had

covered the entire or nearly the entire surface of the

unamended plate; this usually took four days. The colony

diameters were measured in two perpendicular directions

using a metric ruler. The 7-mm diameter of the plug was

subtracted from these values. The effective diameters of

the unamended colonies ranged from 72 to 81 mm. The

average-colony diameter was then calculated.

The average-colony diameter on the amended plate was

divided by the average diameter of the unamended colony.

This value was expressed as a percentage. The percent-

relative growth for an isolate was zero if no mycelia were

observed growing from the plug. The average of two

replicates determined the average-percent-relative growth

for each isolate. These values for each isolate were

averaged to calculate the mean-percent-relative growth for

each plot.

Triadimefon completely inhibited 211 of the 560

isolates tested for this survey (see Table 2.2). Slight

gradations in the degrees of sensitivities were found

among the rest of the isolates. The greatest average-

percent-relative-growth measurements were 3.9% with one

isolate and 3.8% with two. These three isolates were the

57

only ones to have 3.0% or greater average-percent-relative

growth.

None of the isolates were nearly as insensitive as

those reported by Golembieski (30) nor Gilstrap (28) on

isolates collected from turf sites dominated by DM1-

resistant R. floccosum. An analysis of variance (AOV) was

performed that compared the plots where the ensuing

treatments (see below) would be applied. A P-value of

0.595 was insufficient evidence to reject the null-

hypothesis that observations were a result purely of

chance. Therefore, it was concluded that the experimental

site at the HTRC had a R. floccosum population sensitive

to DM1 fungicides.

Fungicide Treatments

Treatment 1 was the control or check treatment with

no fungicides applied to it. The remaining treatments

were different fungicides and timing intervals (see Table

2.3). Changes in the formulations used and their

suppliers changed over the course of the study.

Chlorothalonil applied as Daconil 2787 4.17 FL was

obtained early on from 15K Biotech Co. in Mentor, Ohio,

and later from Zeneca Ag Products in Wilmington, Delaware.

Iprodione (Chipco 26019 2 FL) was supplied by Rhone-

58

Poulenc Ag. Co. in Research Triangle Park, North Carolina.

Triadimefon was provided as Bayleton. Fenarimol (Rubigan

1 AS) was provided by DowElanco of Indianapolis, Indiana.

Propiconazole was applied first as Banner 1.1 EC (Ciba

Co., Greensboro, North Carolina) and later as Banner Maxx

1.3 ME (Novartis, Greensboro, North Carolina).

Treatment 2 received only chlorothalonil, a contact

or multisite (MS) fungicide. Treatment 3 received only

iprodione, a SS fungicide of the dicarboximide class.

Treatments 4, 5, and 7 were triadimefon, fenarimol, and

propiconazole respectively, all at rates and intervals

typically used on dollar spot. Treatment 6 was at the

same propiconazole rate as Treatment 7 but at 14-day

instead of 21-day intervals. Treatment 9 was

propiconazole at four times the typical dollar-spot rate,

which was the maximum specified to control ectotrophic-

root diseases (see below) Treatments 10 and 11 were

intended to test the effects of a DMI- and an MS-fungicide

In combination and alternation respectively. Treatments

12 and 13 were to test the same theory except with a DMI

and a dicarboximide.

Treatment 14 incorporated the use of a DMI applied as

a preventative to suppress summer patch caused by

Magnaporthe poae, Landschoot & Jackson and/or take-all

59

patch caused by Gaeumannomyces graminis (Sacc.) Arx &

Olivo var. avenae (E. M. Turner) Dennis., which are both

ectotrophic-root diseases found on many Michigan golf

courses. The first application of propiconazole occurred

when soil temperatures at a 50-mm depth reached 24 C for

five consecutive days. A second application was then made

28 days later. These annual applications of propiconazole

were followed in years 1 and 4 with propiconazole only,

year 2 with chlorothalonil only, and year 3 with iprodione

only. This treatment simulated the "Yearly Rotation

Theory" recommended by Vargas at the time (85).

Treatments were applied in each of three-sequential-

growing seasons. Treatments 1, 2, 3, 4, and 5 received no

propiconazole. The total number of propiconazole

applications with the other treatments from 1994 through

19~7 ranged from six (treatment 13) to 20 (treatments 6

and 10) as shown in Table 2.4. The total amount of

propiconazole applied to these treatments varied from 25.2

g 100 m-2 (treatments 11 and 13) to 235.2 g 100 m-2

(treatment 9), a greater than nine-fold increase (see

Table 2.5) .

60

Fungicide-Application Techniques

The fungicides were diluted with water and applied

under C02 pressure using a hand-pushed, rolling boom-

sprayer. All passes across the plots with the sprayer

were made with a spray-volume rate of 4.1 I 100 m-2.

Treatment 8 was at two times the propiconazole rates of

Treatments 6 and 7; therefore, two passes were made and

the resulting spray volume applied was equivalent to 8.2 I

100 -2m . The four passes needed to apply the 4 X

propiconazole rates called for in Treatments 9 and 14

resulted in an equivalent spray-volume rate of 16.4 1 100

m-2. The same application method resulted in equivalent

spray-volume rates of 8.2 I 100 m-2 with treatments 10 and

12. Fungicide treatments were initiated in early August

1994 and reapplied according to experimental protocol

until early September.

Sample Collection

The collecting and handling of the samples were the

same as that described above. On 14 October 1994, ten

isolates were taken from each plot if ten infection

centers were present; otherwise, the number of isolates

was the same as the number of infection centers present.

More samples were collected on 14 July 1995. Treatments

61

were then resumed until early September. Dollar-spot

incidence increased until at least ten dollar spots were

present in each plot. Isolates were collected in the same

manner as above on 20 October 1995.

Dollar-spot incidence had been minimal due to

unusually cool weather when a reduced number of samples

were collected on 12 July 1996. Treatments were resumed

on the following day. More favorable conditions for

dollar-spot activity occurred in late summer and early

fall.

Treatments continued through mid October in an

attempt to maximize selection pressure. Isolates were

next collected on 1 August 1997 followed by a resumption

of treatments that continued through mid October. The

next and final sampling occurred on 21 August 1998.

In Vitro Sensitivity Assessments and Statistical Analysis

Ten isolates or fewer from each plot with each

sampling were assayed for DMI resistance as described

above. Ten 4-mm plugs from an unamended plate of each

isolate were stored in individual 1.5 microfuge tubes with

1 ml of mineral oil. An AOV table for each observation

was calculated using Microsoft Office Excel software.

62

Accuracy was checked using the GLM function of SAS and

mlnor errors were corrected on the Excel spreadsheets

Sensitivity Differentiation Using AFLP Markers

A study was conducted in 2002 with Drs. Kurt Lamour,

Brandon Horvath, Mary Hausbeck and Joseph Vargas, Jr.

using the resources of the Hausbeck lab at MSU. The

object of this collaboration was to determine if AFLP

markers could be used effectively to differentiate DMI-

sensitive and DMI-resistant isolates of R. floccosum. Six

isolates already determined to be DMI sensitive and ten

isolates known to be DMI resistant in another study (see

Chapter 3) were analyzed using a commonly used DNA-

extraction and AFLP-fingerprinting methodology described

fully by Lamour (49).

An AFLp™ Microbial Fingerprinting Kit (Perkin-Elmer

Corp., Foster City, California) supplied the PCR-core mix,

core-primer sequences, adaptor sequences, and florescence.

An MJ Research Minicycler (MJ Research Inc., Waltham,

Mass.) was used to perform all PCR reactions. The

selective primers EcoRI-AA, -AC, -AG, and -AT, were used

in all 16 combinations with Msel-CA, -CC, -CG, and -CT

selective primers to perform the PCR amplifications. This

study was of a larger investigation, and further

63

information including a description of the statistical

analysis used to interpret the data has been provided by

Horvath (36).

Results and Discussion

The mean-percent-relative-growth values for each

block and treatment at each observation are shown in

Tables 2.6-2.11. The effective diameters of the unamended

colonies ranged from 70 to 81 mm. Average-percent-

relative growth for any isolate did not exceed 10% until 1

August 1997 when two isolates each measured 10.6% in the

fourth block of Treatments 4 and 10. Isolates in

Treatments 2 and 4 of block 4 measured 14.7 and 27.5

average-percent-relative growth respectively on 21 August

1998. An AOV showed significant differences among

treatments on 20 October 1995 (P < 0.039), 1 August 1997

(P < 0.068), and 21 October 1998 (P <> 0.029). The

relative ranking of treatments on those dates is shown on

Tables 2.12-14.

None of the block or treatment means approached those

that have been found earlier with DMI-resistant

populations of R. floccosum (28,31). Therefore, the

results are best presented and discussed in terms of

relative sensitivities. Several terms will be used as

64

defined below. Mean-percent-relative growth is truncated

to 'relative growth'. The term 'group' denotes a cluster

of treatments separated by values less than the LSD. The

word 'higher' signifies greater R. floccosum sensitivity

to DMls and 'lower' indicates lesser such sensitivity when

comparing two treatments, two groups, or a treatment and a

group. The words 'highest' and 'lowest' designate similar

relationships except that the comparisons involve three or

more groups, treatments, or any combinations thereof. The

use of 'top' or 'bottom' refers to the relative ranking of

any of the above. 'Dollar-spot rate' means the rate or a

rate indicated on the respective-fungicide-product's label

for dollar spot. The words 'observation' and 'date' refer

to those time points having significance. Comparisons on

the first and third dates were made using an LSD value at

p ~ 0.05. Comparisons at the second observation were made

using an LSD value at P ~ 0.10.

Treatment 4 with triadimefon only and Treatment 7

with propiconazole only (see Table 2.3) were in the lowest

group on the first two dates. Both of these treatments

were at dollar-spot rates applied every 21 days.

Treatment 4 was among the second highest group on the last

date. Treatment 6 (propiconazole at the dollar-spot rate

every 21 days) had lower relative-growth values than

65

Treatments 4 and 7 but the differences were not

significant. Treatment 5, (fenarimol at the dollar-spot

rate every 21 days) was the highest ranking member of the

lowest group on the first observation and of the second-

lowest group on the second date. Propiconazole treatments

at higher rates every 21 days (Treatments 8 and 9) were

ranked below all of the above treatments on all dates but

not different than them. These trends lend credence to

the principle that as DMI resistance increases in a

population of R. floccosum, higher rates and/or shorter

intervals of a DMI fungicide are needed to achieve the

same results seen earlier with the same fungicide when the

population was DMI sensitive.

Treatment 10 (propiconazole at the same rate as

Treatments 6 and 7 plus chlorothalonil on 14-day

intervals) was ranked in the second-lowest group and among

the top group on the first date. Treatment 10 had the

highest ranking at the second and third observations.

These measurements increased almost 50% between the first

and second date and 35% between the second and third dates

representing a two-fold difference between the first and

third dates (see Figure 2.2).

Chlorothalonil alone every 14 days (Treatment 2) was

among the treatments in the higher group as well as the

66

lower group on the first dates. It was In the higher

group and not different than Treatments 4, 7, and 10 on

the second date. It was In the highest group and not

different than Treatment 10 at the last observation where

it was also at the top of the second highest group on the

last observation and not different than Treatments 4, 5,

6, 7, 11, and 14.

Increased R. floccosum sensitivities to the DMls over

time in treatments that utilized chlorothalonil have been

noted in earlier small-plot studies by Golembieski et al.

(31) and Gilstrap et al. (28). Both phenomena were

explained by the possible occurrence of migration events

of resistant fungi from adjacent or nearby DMI-treatment

plots. However, chances of such influx were lessened here

because this large-plot experiment was designed to

minimize plot-to-plot movement of diseased clippings (see

Figure 2.1).

Isolates with decreased DMI sensitivities were

detected in Block 4 of Treatment 2, the chlorothalonil

only treatment, on 1 August 97 (see Table 2.10). Isolates

taken the next year had similar levels of resistance

except for one that had an average-percent-radial-growth

value of 14.7, which was next to greatest value detected

at that observation or in the experiment, overall. This

67

isolate could have been the result of a mutation that had

occurred or it could have been a migrant, which was

unlikely (see above). As noted, other isolates with

decreased DMI sensitivities were detected. This leads to

speculation that this subpopulation underwent

physiological changes during this experiment. One such

hypothesis is that repeated exposures to the MS-fungicide

chlorothalonil can enhance R. floccosum resistance to DMls

and perhaps other SS-fungicide classes.

Pujls and Shaw made single applications of the DMI

flutriafol and chlorothalonil each alone and ln

combination or ln alternation to three successive

plantings of wheat each having a DMI-sensitive population

of Septoria tritici Rob. ex Desm., the causal organism or

wheat blotch. In vitro differences in DMI tolerances were

not found to be significant. However, a full rate of

flutriafol combined with a full rate of chlorothalonil

showed the greatest shifts towards less sensitive isolates

(62 )

Koller and wilcox reported increased resistance to

both the DMls and benzimidazoles in Venturia inaequalis

(Cook) G. populations already resistant to dodine, also an

MS fungicide (25), as compared to those dodine sensitive

(47) . The greatest and most significant increases to both

68

the DMls and benzimadazoles occurred with those isolates

that were already multiresistant to both classes prior to

further exposures.

Dodine was one of the first MS fungicides to which

field resistance developed (82). Anilazine is also an MS

fungicide, and resistance to it by R. floccosum was

reported by Nicholson et al. in 1971 (60). Reduced

sensitivity of Colletotrichum gloeosporioides (Penz.)

Penz.&Sacc. to the MS fungicide chlorothalonil has been

shown in vitro (48).

Koller and wilcox speculated that a membrane-

transport system enabled the active outflux of unrelated

fungicides and could have been responsible for their

findings (47). Such physiology has been suggested as a

possible mechanism that could facilitate such

multiresistance (17,19). Multidrug resistance (MDR) has

been linked to an increased expression of transporter

genes that produce transporter proteins belonging to the

ubiquitous superfamily of ATP-binding cassette (ABC)

transporters (2), which is one of the largest known

protein classes (51).

These proteins use energy produced Vla ATP hydrolysis

to traffic molecules across biological membranes (81).

Single proteins can facilitate a diversity of substrates

69

(83). They are known to confer MDR to anti-tumor drugs

(37) and are a major concern for cancer therapy (70). An

over-expression of some of these transporters has been

linked to MDR in laboratory-induced mutants of filamentous

fungi (2,80). These phenomena have also been documented

with field isolates of Botrytis cenerea (50) and

Mycosphaerella graminicola (81). It is conceivable that

ABC transporters in R. floccosum contributed to the

pathogen's increased-DMI sensitivity shown with Treatment

2 and Treatment 10, both of which involved sequential

applications to chlorothalonil, compared to other

treatments as described above.

Treatment 11 that alternated propiconazole and

chlorothalonil was among the lowest groups at all three

observations. This was most likely because of the fewer

number of exposures as well as the overall lower amount of

the DMI applied compared to the other propiconazole

treatments, and perhaps the other chlorothalonil

treatments as well (see Tables 2.4 and 2.5). These

results support the alternation of DMIs with

chlorothalonil as an effective strategy to delay DMI

resistance.

The treatments using iprodione (Treatments 3, 12, and

13) also ranked similarly at the bottom. Implementing one

70

or more of these four treatments into a dollar-spot

management program appears to have merit. However,

questions concerning R. floccosum resistance to iprodione

and whether or not it occurred in this experiment are

valid. That issue was beyond the scope of this

experiment, but it could be investigated as long as the

isolates from those iprodione plots could be grown again

from stored samples.

The treatment effects with the yearly-rotation theory

are difficult to interpret in relation to the other

treatments since it was the only one having two DMI

applications annually prior to the advent of dollar-spot

treatments. The fact that it was among the second and

third groups on the second and third observations is

reason to consider its utility. An attribute is that it

allows three-year intervals between iprodione treatments.

This respite could allow time during which dicarboximide-

sensitive could regain dominance as the dicarboximide-

resistant isolates fail to survive due to their poor

environmental fitness (32,52). How the results would have

changed had the other treatments included early DMI

applications were not answered due to area and resource

limitations of this experiment.

71

The fewer numbers of isolates collected in Treatment

10 in the third and fourth blocks at the last observation

were a result of fewer dollar spots being present at the

time when a full contingent of isolates were collected

from 46 of the 54 plots. This suggests that the repeated

applications of propiconazole in combination with

chlorothalonil eliminated sensitive strains. This would

reduce the competition in the infection court so that a

resistant strain could begin to flourish. This process

appears to have started in Block 4 and to a lesser degree

in block B in Treatment 10. This reasoning suggests that

a shift could have begun in Treatment 12 since only four

isolates were collected in each of its blocks as well as

In block 2 of Treatment 6.

Burpee challenged two propiconazole-sensitive strains

to series of 10 in-vitro exposures to propiconazole

concentrations of 0.2 pg ml-1 and 0.3 pg ml-1 and observed

only slight but significant (P ~ 0.05) decreases in

sensitivities that did not approach nearly the lack of

sensitivity demonstrated by an isolate known to be DMI

resistant (15). De Waard and Van Nistelrooy also showed

in vitro loss of sensitivity with penicillium italicum

Wehrner. to DMls using similar methodology (18). Results

presented here are evidence that repeated applications of

72

DMls to field populations of R. floccosum may cause

similar decreases in DMI sensitivity in planta.

The reduced DMI sensitivity with Treatment 10 on 1

August 1997 (see Table 2.13) occurred after 14

applications of a DMI (see Table 2.4). The further

reduction on 21 August 1998 followed six more DMI

applications. This evidence supports Vargas' estimate

that between 20 and 30 applications of a DMI can be made

before resistant isolates begin to emerge (86,87).

Opportunities to extend this range may exist by limiting

the long-distance, inward migration of leaf tissue, some

of which could influx DMI-resistant isolates of R.

floccosum. This may be particularly true for new golf

courses where the turf was established solely from seed.

Greater attention toward the sanitation of golf clubs,

golf shoes, and turf-maintenance equipment that comes from

other golf courses may be strategically beneficial.

The goals of this study could probably have been

addressed more efficiently if each plot's sampling area

had been inoculated initially and equally with a DMI-

resistant strain. A suitable amount of inoculum would

have been that needed to be detected in approximately 20%

of the baseline samples. Also, the use of a single DMI at

its respective dollar-spot rate would have reduced the

73

total number of treatments. An example of such a

treatment regime is shown in Table 2.16.

The AFLP analysis was inconclusive. The markers used

were linked to random base pairs and did not provide

adequate resolution. Identification of a DNA sequence or

sequences that could be used to differentiate DMI-

resistant and DMI-sensitive R. floccosum would have been a

serendipitous discovery. However, it might be attained

uSlng additional sets of markers different than those used

here. Comparisons of radial growth on PDA only and PDA

amended with fungicides continue to be the most efficient

and discriminate method for assessing the relative-

fungicide sensitivities of R. floccosum strains.

The experimental objective of determining if a DMI-

sensitive population of R. floccosum could be shifted to a

predominantly DMI-resistant population by the directional-

selection pressure of repeated applications of a DMI

fungicide was not achieved. However, a population with

reduced sensitivity was detected following repeated

applications of propiconazole when combined with

chlorothalonil (Treatment 10). This occurred after 20

applications of a DMI. Determining this number of

applications was an alm of this study.

74

Reduced DMI sensitivity with the R. floccosum

population that occurred with Treatment 10 was greater

than that detected when propiconazole was alternated with

chlorothalonil (Treatment 11). This finding addressed one

of the questions concerning the effects of a DMI combined

with a fungicide of a different class, or a DMI alternated

with such.

The R. floccosum populations associated with several of

the other DMI treatments appeared to be on the verge of

shifting toward reduced DMI sensitivities when the

experiment was terminated. The relative lack of dollar-

spot incidence ~n some of those treatments indicated that

the common strains of R. floccosum were being eliminated

due to repeated exposures to a DMI fungicide. It IS

suspected that significant reductions in the DMI

sensitivities of these populations would have occurred had

the experiment been extended for several more years.

75

Table 2.1. Common names, chemical names, trade names, andcurrent registrants of demethylation-inhibitor fungicideslabeled for use on commercial turf in the U. S.

Fungicide VII Andersons1% Bayleton

Common Name

triadimefon

Chemical Name

1-(4-chloro-phenoxy)-3,3-dimethyl) -1- (lH-1,2,4-triazol-1-yl)-2-butanone

Trade Name

Bayleton 50

Granular TurfFungicide

Regi-strant

fenarimol Q-(2-chlorophenyl) -Q-(4-chloro-phenyl)-5-pyrimi-dine-methanol)

propiconazole 1-[[2(2,4-dichlo-rophenyl)-4-propyl -1,3-dioxolan-2-yl]methyl]l-H-1,2,4-triazole)

myclobutanil 2-(4-chloro-phenyl)-2-(lH-1,2, 4-triazol-1-yl methyl)hexane-nitrile

Accost Verdicon4

Rubigan A.S. Gowan5

Twosome Lesco

Banner GL syngenta6

Banner Maxx

Spectator Lesco

Propiconazole BASF7

ProEagle Dow8

Manhandle Lesco

Golden Eagle Andersons

lBayer CropScience, Kansas City MO2Lesco, Inc., Rocky River OH3Andersons Golf Products, Maumee OH4Verdicon, Inc., Sioux Falls IA5Gowan Company, Yuma AZ6Syngenta Professional Products, Greensboro NC7BASF Professional Turf, Research Triangle Park NCsDow AgroSciences, Indianapolis IN

76

Table 2.2. Average-percent-relative growth for each blockof ten subsamples collected on 29 July 1994 and mean-percent-relative growth for each treatment shown alongwith the fungicides for each treatment.

TreatmentX

1. control

2. chlorothalonil (95.5)

3. iprodione (15.3)

4. triadimefon (7.6)

5. fenarimol (6.7)

6. propiconazole (4.2)




10. propiconazole (4.2) +chlorothalonil (95.5)

11. chlorothalonil (95.5) alt.wi propiconazole (4.2)

12. propiconazole (4.2) +iprodione (15.3)

13. iprodione (15.3) alt.wi propiconazole (4.2)

14. Yr 1 and 4propiconazole (16.8)propiconazole (4.2)

Yr 2propiconazole (16.8)chlorothalonil (95.5)

Yr 3propiconazole (16.8)iprodione (15.3)

XRates in 9 AI 100 m-2.

YIntervals between applications.

77

DaysY I II III IV Mean

0.91 0.89 1.17 0.77 0.94

14 1.17 0.89 1. 02 1. 32 1.10

21 0.75 0.76 0.50 0.65 0.67

21 0.64 0.90 0.41 0.76 0.68

21 0.78 0.37 0.77 0.91 0.71

14 1. 28 0.88 0.88 1. 03 1. 02

21 1. 68 0.52 1. 03 1. 03 1. 06

21 1. 02 1. 40 0.90 1. 28 1.15

21 0.78 1. 43 0.63 0.75 0.90

14 1. 01 0.64 1.15 0.77 0.89

14 alt. 0.00 1. 30 1. 28 1. 39 0.99wi 21

21 0.63 0.75 0.91 1.18 0.87

21 0.64 1. 64 0.89 0.77 0.99

24C&28d 0.65 0.89 1.27 1.15 0.9928

24C&28d14

24C&28d21

Table 2.3. Application rates and timing intervals of treatments using one or more fungicides as shown.

Treatment Rate (g 100 itf2) Interval (days)

1. control

2. chlorothalonil 95.5 14

3. iprodione 15.3 21

4. triadimefon 7.6 21

5. fenarimol 6.7 21

6. propiconazole 4.2 14




10. propiconazole + 4.2+95.5 14 chlorothalonil

11. chlorothalonil alt. 95.5 alt. w/ 4.2 14 alt. w/ 21 w/ propiconazole

12. propiconazole + 4.2+15.3 21 iprodione

13. iprodione alt. 15.3 alt. w/ 4.2 21 w/ propiconazole

14. Yr 1 and 4 propiconazole propiconazole

Yr 2 propiconazole chlorothalonil

Yr 3 propiconazole iprodione

16.8 4.2

16.8 95.5

16.8 15.3

at

at

at

24C & 28d 28

24C & 28d 14

24C & 28d 21

78

Table 2.4. Number of propiconazole applications appliedin 1994, 1995, 1996, and 1997.

1994

1995

1996

1997

Total

Tmt. 13& 11

1

1

2

2

6

Tmt. 14

3

2

2

3

10

Tmts. 7a,8, 9, & 12

3

3

4

4

14

Tmts. 6& 10

4

4

6

6

20

aTriadimefon and fenarimol treatments were applied atequivalent rates on the same days as Trot. 7.

Table 2.5. Propiconazole amounts in g 100-2 applied in1994, 1995, 1996, and 1997.

Tmts. 11& 13

Tmts. 7,8, & 12

Tmts.6 & 10

Trot.14

Tmt. 9

1994 4.2 12.6 16.8 42.0 50.4

1995 4.2 12.6 16.8 37.8 50.4

1996 8.4 16.8 25.2 37.8 67.2

1997 8.4 16.8 25.2 42.0 67.2

Total 25.2 58.8 84.0 159.6 235.2

79

Table 2.6. Average-percent-relative growth for each blockof ten subsamples (or fewer where noted) collected on 14October 1994 and mean-percent-relative growth for eachtreatment shown along with the fungicides for eachtreatment.

TreatmentX

1. control


3. iprodione (15.3)


5. fenarimol (6.7)












XRates in g AI 100 m-2•

YIntervals between applications.

aNine subsamples collected.

bEight subsamples collected.

80


1. 01 0.51 1.17 1.15 0.96

14 0.51 1.040.81b 0.53 0.72

21 1. 05 0.25 0.64 0.38 0.58

21 0.90 0.77 1. 05 0.79 0.88

21 1. 03 0.90 0.90 1. 42 1. 06

14 1.17 0.75 1. 03 0.77 0.93

21 0.52 0.77 0.52 0.79 0.65

21 1. 41 1. 39 0.63 1. 04 1.11

21 0.90 1. 28 0.90 0.78 0.97

14 1. 05 1. 05 0.76 1.13 1. 00

14 al t. 0.25 1. 00 1.13 1.14 0.88wi 21

21 0.78 1. 27 1. 02 1. 43" 1.13

21 0.78 1. 03 1. 28 1. 45 1.14

0.29" 1. 42 1. 04 1. 56 1. 0824C&28d

28

24C&28d14

24C&28d21

Table 2.7. Average-percent-relative growth for each block of ten subsamples (or fewer where noted) collected on 14 July 1995 and mean-percent-relative growth for each treatment shown along with the fungicides for each treatment.

Treatment Daysy II III IV Mean

1. control


3. iprodione (15.3)


5. fenarimol (6.7)





10. propiconazole (4.2) + chlorothalonil (95.5)

11. chlorothalonil (95.5) alt. w/ propiconazole (4.2)

12. propiconazole (4.2) + iprodione (15.3)

13. iprodione (15.3) alt. w/ propiconazole (4.2)

14. Yr 1 and 4 propiconazole (16.8) propiconazole (4.2)

Yr 2 propiconazole (16.8) chlorothalonil (95.5)

Yr 3 propiconazole (16.8) iprodione (15.3)

xRates in g 100 m"2. YIntervals between applications.

Nine subsamples collected.

Eight subsamples collected.

1.15 0.75 1.18 0.75 0.96

14 0.65 0.65 1.30 1.60 1.05

21 0.64 0.78 0.64b 0.78 0.71

21 1.28 1.04 1.18 1.17 1.17

21 0.90 0.13 1.29 1.14 0.87

14 0.63 0.90 1.01 1.54 1.02

21 0.89 1.16 0.52 0.39 0.74

21 0.91 1.03 0.90 1.01 0.96

21 1.13 0.76 1.26 0.51 0.91

14 0.91 1.78 0.63 2.06 1.35

14 alt. 0.39 1.03 0.91 0.38 0.68 w/ 21

21 1.29a 1.55 1.42 0.57 1.21

21 0.77 0.90 1.55 0.64 0.96

0.57 1.66 1.16 1.15 1.14a

24C&28d 28

24C&28d 14

24C&28d 21

81

Table 2.8. Average-percent-relative growth for each blockof ten subsamples collected on 20 October 1995 and mean-percent-relative growth for each treatment shown alongwith the fungicides for each treatment.

TreatmentX DaysY I II III IV Mean

1. control2. chlorothalonil (95.5)3. iprodione (15.3)4. triadimefon (7.6)5. fenarimol (6.7)6. propiconazole (4.2)



9. propiconazole (16.8)10. propiconazole (4.2) +

chlorothalonil (95.5)







XRates in g 100 m-2•

Ylntervals between applications.

82

0.38 1. 01 0.77 1.16 0.8314 0.79 0.50 0.51 2.00 0.9521 0.64 0.26 0.53 1. 30 0.6821 0.76 1. 96 1. 56 1. 06 1. 3321 0.78 1. 66 1. 53 1. 93 1. 4714 1. 25 1. 84 0.53 1. 65 1. 32

21 1. 55 0.51 1. 03 2.56 1. 41

21 1. 54 0.65 0.89 1.16 1. 06

21 0.52 1. 41 1. 01 1. 91 1. 2114 1.16 0.89 1. 56 1. 94 1. 39

14 alt. 0.51 0.77 0.38 1.13 0.70wi 21

21 0.65 1. 04 0.51 0.78 0.74

21 0.64 0.91 0.66 0.51 0.68

1.28 1.78 0.78 1. 54 1. 3424C&28d

28

24C&28d14

24C&28d21

Table 2.9. Average-percent-relative growth for each blockof ten subsamples collected on 12 July 1996 (or fewerwhere noted) and mean-percent-relative growth for eachtreatment shown along with the fungicides for eachtreatment.

TreatmentX DaysY I II III IV Mean

l. control 0.44 g 2.87t 1. 66

2. chlorothalonil (95.5) 14 1.35t 1. 35

3. iprodione (15.3) 21 0.93t5.62t4.01t1.2ge 2.97

4 . triadimefon (7.6) 21 1.28g 1. 42d 1. 35

5. fenarimol (6.7) 21 1.39b2.34e 1. 87

6. propiconazole (4.2) 14 1.33eO.48g2.94a3.12t 1. 97

7. propiconazole (4.2) 21 2.20t 1. 75d 4. 94g 2. 93b 2.96

8. propiconazole (8.4) 21 2. SOh1 .71 d 1 . 36d 4 . 40c 2.49

9. propiconazole (16.8) 21 1.64c 1.17t2.42e 1. 74

10. propiconazole (4.2) 14 1.83e3.6ge1.83c2.51c 2.47chlorothalonil (95.5)



14 alt. 1.38d2.47t1.33dO.63t 1.45wi 21





21

24C&28d28

24C&28d14

24C&28d21

XRates in 9 100 m-2.

Ylntervals between applications.aNine subsamples collected.

bEight subsamples collected.cSeven subsamples collected.

83

dSix subsamples collected.epive subsamples collected.fpour subsamples collected.

gThree subsamples collected.hTwo subsamples collected.

Table 2.10. Average-percent-relative growth for eachblock of ten subsamples collected (or fewer where noted)on 1 August 1997 and mean-percent-relative growth for eachtreatment shown along with the fungicides for eachtreatment.

TreatmentX

1. control2. chlorothalonil (95.5)3. iprodione (15.3)4. triadimefon (7.6)5. fenarimol (6.7)6. propiconazole (4.2)


8. propiconazole (8.4)9. propiconazole (16.8)








XRates in 9 100 m-2•

Ylntervals between applications.aNine subsamples collected.

84


1. 06 1. 07 0.75 0.59" 0.8714 0.84 1. 34 1. 51 2.98 1. 6721 1.16" 1. 27 0.85 0.75"1.0121 0.89 0.74 1. 73 2.93 1. 5721 1. 05 0.63 2.06 1.40 1. 2914 1. 93 1. 60 1. 76 0.84 1. 53

21 2.05 1.16 2.19 1. 51 1. 73

21 0.92 1.14 1. 48 1. 60 1. 2921 0.90 1. 23 1. 69 0.87" 1.1814 0.79 1. 99 2.19 3.84 2.20

14 alto 0.79 0.99 1. 39 1. 39 1.14wi 21

21 1. 08 0.79 1.07 0.66b 0.90

21 0.62 0.62 0.99 1.05" 0.82

24C&28d 1.231.031.511.02" 1.2028

24C&28d14

24C&28d21

bEight subsamples collected.cSeven subsamples collected.

Table 2.11. Average-percent-relative growth for eachblock of ten subsamples collected (or fewer where noted)on 21 August 1998 and mean-percent-relative growth foreach treatment shown along with the fungicides for eachtreatment.

Trea trnentX DaysY I II III IV Mean

1. control2. chlorothalonil (95.5)3. iprodione (15.3)4. triadirnefon (7.6)5. fenarirnol (6.7)6. propiconazole (4.2)








0.86 0.86 0.50 1.00 0.8114 1.11 1. 42 1. 29 4.15 1. 9921 0.86 0.42 0.80 0.75 0.7121 0.37 0.90 0.64 4.66 1. 64

21 0.86 0.50 1. 28 3.15 1. 45

14 2.480.97d 1.13 0.63 1. 30

21 1. 01 0.77 1.21 3.13 1. 53

21 0.76 0.26 1. 00 1. 27 0.82

21 1. 38 0.25 0.25 1. 36 0.81

14 2.02 2.702.53b4.60c 2.96

14 al t. 1. 00 1. 261. 45a 1. 89 1. 40wi 21

21 0.78 0.80 0.62 0.75 0.74

21 0.88 0.13 0.00 2.89 0.97




24C&28d28

24C&28d14

24C&28d21

=================:::::;:b============XRates in g 100 rn-2• Seven subsarnples collected.Ylntervals between applications. cSix subsarnples collected.aEight subsarnples collected. dFour subsarnples collected.

85

Table 2.12. Treatment means of percent-relative-growthvalues of four replications collected on 20 October 1995.

No. TreatmentRate

(g 100 m-2)Interval

(days)Mean-

RelativeGrowth

5. fenarimol10. propiconazole +

chlorothalonil

7. propiconazole4. triadimefon

14. Yr 1 and 4propiconazolepropiconazole

Yr 2propiconazolechlorothalonil

Yr 3propiconazoleiprodione

6. propiconazole9. propiconazole8. propiconazole2. chlorothalonil1. control

12. propiconazole +iprodione

11. chlorothalonilalt. wipropiconazole

3. iprodione13. iprodione alt.

wi propiconazole

6.74.2 +95.5

4.27.6

16.84.2

16.895.5

16.815.3

4.216.88.4

95.5

4.2 +15.3

95.5 altowi 4.2

15.315.3 alt.wi 4.2

LSD at

2114

2121

at 24C &28d, 28

at 24C &28d, 14

at 24C &28d, 21

14212114

21

14 al t.wi 21

2121

p S; 0.05

1.48

1.40

1.40

1.38

1.35

1.30

1.20

1.08

0.950.850.73

0.70

0.680.68

0.62

AB

ABABABCD

ABCDEABCDEABCDEABCDEBCDECDE

E

E

E

-=xMeans followed by the same letter are not significantly differentfrom each other.

86

Table 2.13. Treatment means of percent-relative-growthvalues of four replications collected on 1 August 1997.

No. TreatmentRate

(g 100 m-2)Interval

(days)

Mean-RelativeGrowth

10. propiconazole +chlorothalonil

7. propiconazole2. chlorothalonil4. triadimefon6. propiconazole5. fenarimol8. propiconazole

14. Yr 1 and 4propiconazolepropiconazole



9. propiconazole11. chlorothalonil

alt. wipropiconazole

3. iprodione12. propiconazole +

iprodione

1. control13. iprodione alt.

wi propiconazole

4.2 +95.5

4.295.57.64.26.78.4

16.84.2

16.895.5

16.815.3

16.895.5 alt.wi 4.2

15.34.2 +15.3

15.3 al t.wi 4.2

LSD at

14

211421142121

at 24C &28d, 28

at 24C &28d, 14

at 24C &28d, 21

2114 alt.wi 21

2121

21

p ~ 0.10

2.20

1.73

1. 67

1. 57

1. 53

1. 45

1.29

1.20

1.18

1.14

1. 01

0.90

0.870.82

0.66

AB

ABCABCBCDBCDEBCDEBCDE

BCDEBCDE

CDEDE

E

E

XMeans followed by the same letter are not significantly differentfrom each other.

87

Table 2.14. Treatment means of percent-relative-growthvalues of four replications collected on 21 August 1998.

No. TreatmentRate

(g 100 m-2)Interval

(days)Mean-

RelativeGrowth

10. propiconazole +chlorothalonil

2. chlorothalonil4. triadimefon7. propiconazole5. fenarimol

11. chlorothalonilalt. wipropiconazole

6. propiconazole14. Yr 1 and 4

propiconazolepropiconazole



13. iprodione alt.wi propiconazole

8. propiconazole9. propiconazole1. control

12. propiconazole +iprodione

3. iprodione

4.2 +95.5

95.57.64.26.7

95.5 alt.wi 4.2

4.2

16.84.2

16.895.5

16.815.3

15.3 alt.wi 4.2

8.416.8

4.2 +15.3

15.3

LSD at

14

14212121

14 alt.wi 21

14

at 24C &28d, 28

at 24C &28d, 14

at 24C &28d, 21

21

2121

21

21

p ~ 0.05

2.96

1. 99

1. 64

1. 53

1. 45

1.40

1. 30

1. 22

0.97

0.820.810.810.74

0.71

1.11

ABBCBCBCBC

BCBC

BC

C

C

C

C

C

XMeans followed by the same letter are not significantly differentfrom each other.

88

Table 2.15. Number of chlorothalonil applicationsapplied in 1994, 1995, 1996, and 1997.

Year

1994

1995

1996

1997

Total

Trot. 11(propiconazole alt.wi chlorothalonil)

2

2

3

3

10

89

Trot. 2(chlorothalonil alone)

and Trot. 10(propiconazole +chlorothalonil)

4

4

6

6

20

Table 2.16. Chlorothalonil amounts In g 100 m-2 applied In1994, 1995, 1996, and 1997.

Year

1994

1995

1996

1997

Total

Tmt. 11(propiconazole alt.wi chlorothalonil)

191

191

287

287

955

90

Tmt. 2(chlorothalonil alone)

and Trot. 10(propiconazole +chlorothalonil)

382

382

573

573

1910

Table 2.17. An example of application rates and timingintervals of treatments for a study that would have hadgreater efficiency than the one conducted and presented InChapter 2.

Treatment Rate (g 100 m-2) Interval (days)

1. control

2. chlorothalonil 45.3 10 alt 11


4. iprodione 15.3 21

5. propiconazole + 4.2 + 45.3 21chlorothalonil

6. propiconazole alt. 4.2 alt. 21 alt. wiwi chlorothalonil wi 45.3 10 or 11

7. iprodione + 15.3 + 45.3 21chlorothalonil

8. iprodione alt. 15.3 alt. 21 alt. wiwi chlorothalonil wi 45.3 10 or 11

9. propiconazole + 4.2 + 15.3 21iprodione

10. propiconazole alt. 4.2 alt. 21wi iprodione wi 15.3

91

12 14 3 8 11 9 1

N

r - - --I r----. r----' r----. r---- r - - --, r----I I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I , I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I I,,--- -" "----,, 1.----" 1.----" ,,---- ,,-- --" "'----

r---- r---- r - - --. r----. r -- --I r - - --. r----.I I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I II I I I I I I I I I I I.. ---- 1.-- __ .. ----" 1. ____ 01 1.--- _" 1.----" ,,--- - ..

6 4 2 7 10 5 13

Figure 2.1. Map of Block 4 with treatment numbersabove or below each 2.1 m by 8.4 m (17.6 m2) plot with1.2 m by 3 m (3.6 m2) sampling areas outlined andmowing directions indicated by parallel arrows lnopposite directions.

92

3.5

A3

2.5

2

1.5

1

0.5

o20 Oct 95P ~ 0.05

1 Aug 97p ~ 0.10

21 Aug 98p ~ 0.05

~ Tmt 11DTmt 2• Tmt 10

- chlorothalonil alt. propiconazolechlorothalonil- propiconazole + chlorothalonil

XTreatments sharing the same letter are not significantlydifferent.

Figure 2.2. Mean-percent-relative growth of threetreatments with four replications at three-observationdates with each of the treatments that appliedchlorothalonil.

93

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