CHAPTER I

IDENTIFICATION, CHARACTERIZATION ANDHOST RANGE OF THE PATHOGEN OF

LEAF BLIGHT AND CROWN ROT

6

INTRODUCTION

Original isolations of the fungus from the blighted 'Toronto'

creeping bentgrass greens and the proof of its pathogenicity were made

in 1977 (Larsen et aI, 1980). However, only a brief justification for

the identification of the pathogen as Drechslera catenaria (Drechs.)

Ito (syn. Helminthosporium catenarium) was reported. The information

included here confirms that report and provides additional information

regarding the host range and character of D. catenaria.

Conidial characters are commonly used to separate the Drechslera

species since the perfect state of many species has not been identified.

Several Drechslera species have conidial morphologies that appear

similar and dimensions that often overlap. The original descriptions of

the conidia of Q. catenaria, D. dictyoides, D. erythrospila, Q. phlei,

D. poae and Q. siccans have been listed in the literature (Drechsler,

1923; Drechsler, 1935; Graham, 1955) and distinctions between species

have been made in various taxonomic keys (Luttrell, 1951; Nisikado,

1928; Shoemaker, 1959). Most Drechslera species have cylindric conidia

that are nearly as wide at the apical septum as at the basal septum

(Shoemaker, 1962). For example, Q. erythrospila, Q. poae and Q. siccans

have cylindrical spores that taper slightly toward both ends with the

widest point usually in the middle. Only three species of 'Drechslera

have conidia that taper to a narrower apex: Q. 'catenatia, Q. dictyoides

7

8

and Q. phlei. The widest point of these tapered spores is commonly

at or near the base. The conidia of Q. dicty6ides are widest near the

basal septum, but conidia of Q~ catenatia and Q~ ·phlei are widest near

the second or third cell from the base (Shoemaker, 1962; Chidambaram·et aI,

1973).

The conidia of Q. catenaria are the longest of all the species

discussed herein; however, smaller more irregularly shaped conidia,

possibly immature, appear to be common to all Drechslera species

(Drechsler, 1923; Braverman and Graham, 1960; Graham, 1955; Shoemaker,

1962; Wilkins, 1973). Q. catenaria produces secondary conidia which

develop from a septate apical prolongation of the primary conidium

resulting in chains of two to five (Drechsler, 1923). Drechslera phlei

has also been reported to produce conidia in chains, either with or

without the short interveningconidiophores (Shoemaker, 1962; Graham,

1955). The original description of Q. dictyoides does not report this

production of secondary conidia (Drechsler, 1923). However, some

researchers indicate the formation of catenate conidia (Chidambaram,

1973; Shoemaker, 19~2) by Q. dictyoides, while others do not (Wilkins,

1973; Braverman and Graham, 1960). Shoemaker (1962) reported that

secondary conidia occur regularly in Q. catenaria and Q. phlei but may

occur in other species that have been subjected to prolonged incubation

at high humidity.

The basal cells of Q~ ·phlei and Q. catenatia are longer than wide,

being herni-ellipsoidal in shape (Drechsler, 1923; Shoemaker, 1962;

Chidambaram, 1973). The basal cells of conidia are shorter and broader

in D. dictyoides and are considered hemi-spherical (Drechsler, 1923).

9

This rounded basal cell has also been reported to appear inflated with

a constriction at the first septum of the conidium, a character

visible under the stereobinocular microscope (Chidambaram, 1973;

Morrison, 1980). Dtechslera phlei. conidia are abruptly enlarged at the

second cell from the base while D. catenaria conidia have a smooth

contour without enlarged cells or constrictions of septa (Shoemaker,

1962). All three species contain the hilum within the contour of the

basal cell.

Size of conidia and number of septa per conidium continue to be

important in the separation of Drechslera species. However, size and

shape of conidia in this group of fungi have been shown to be affected

by the culture medium, temperature, light and age of the culture

(Elliott, 1949; Harding, 1975; Kafi and Tarr, 1966; Ruppel, 1974;

Shoemaker, 1962; Tarr and Kafi, 1968; Vargas and Wilcoxson, 1969).

The initial pH of the culture medium was also found to affect conidial

morphology (Harding, 1975; Tarr and Kafi, 1968). Ruppel (1974) reported

differences in conidial dimensions, especially length, when the

Drechslera state of Cochliobolus spicifer (=Bipolaris victoriae sensu

Shoemaker, 1959) was grown on five different culture media. However,

conidia produced on necrotic tissue were longer than those from potato

dextrose agar. Shoemaker (1959) reported that temperature and culture

age also affected conidial dimensions, but light had no effect. The

selection of the mounting fluid prior to conidium observation might

also have an effect on these characters, but Ruppel (1974) found that

conidia mounted in distilled water, lactophenol or Shear's mounting fluid

did not differ in dimensions.

10

Characteristics of fungi in culture may be of considerable value

in distinguishing species. Braverman and Graham (1960) observed the

production of many sclerotia-like bodies, sometimes referred to as

protothecia, in potato dextrose agar cultures of Q. "dictyoides from

tall fescue and D. erythrospila but not in cultures of Q. catertaria,

Q. phlei or the D. dictyoides from ryegrass. Q. dictyoides has also

been reported to produce small spherical sclerotia on 2% malt agar

(Luttrell, 1951). Drechslera dictyoides from tall fescue produced

protothecia in and on sucrose proline agar while Q. erythrospila

produced them mostly on the surface (Shoemaker, 1962). However,

Shoemaker (1962) suggested that protothecia production probably varies

with the culture medium.

Certain Drechslera species have been reported to produce pigments

in the culture medium, when grown on sucrose proline agar in the dark

(Shoemaker, 1962). Using this technique with fresh monoconidial

isolates, Shoemaker found consistent pigment production: yellow in

D. catenaria, brown in Q. erythrospila and Q. fugax, and pink in

D. dictyoides and D. siccans. Pigment production has been observed

and studied by Charles et al (1933) and Raistrick et al (1934) who

were able to purify and identify some of these pigments. They reported

that D. catenaria produced catenarin, a hydroxyanthraquinone which is

orange.

Among Drechslera species, there are varied pathogenicity reports.

D. phlei was reported to only affect timothy (Phleum ptatense)

(Graham, 1955). D. dictyoides was originally reported on Festuca spp.

11

(Drechsler, 1923) but other workers have reported severe blighting of

Lolium spp. and Poa pratensis by this fungus (Braverman and Graham,

1960; Couch, 1973). Morpho~ogic, physiologic and pathologic studies of

Q. dicty6ides by Braverman and Graham (1960) resulted in separation of

two physiologic forms of this fungus: Q. dictyoides f. sp. dictyoides

from meadow and tall fescue and Q. dicty6ides f. sp. perenne from

perennial and annual ryegrasses. Drechslera erythrospila was originally

described as causing red leaf spot (Drechsler, 1935) and has only been

reported on Agrostis spp.: ~. alba, A. palustris, ~. perennans,

~. spica-venti, A. stolonifera and A. tenuis (Couch, 1973; Shoemaker,

1962; V.S.D.A., 1960). Q. catenaria, as previously described, causes

blighting of various forage grasses. However, Q. erythrospila is the

only Drechslera species discussed that has been reported to cause a

disease of Agrostis palustris.

Although differences in conidial morphology are reported in the

published descriptions of Drechslera species, some species are not

quickly or easily identified. Labruyere (1977) reflected this difficulty

and made no distinction between D. catenaria and Q. dictyoides when

identifying fungi on incubated ryegrass seed. Therefore, it seemed

necessary to compare the fungal isolate from bentgrass with the herbaria

type specimens to insure proper identification. In this investigation,

the morphology, physiology and pathogenicity of the Drechslera isolate

from 'Toronto' creeping bentgrass are compared to the characters of

other related Drechslera species and are used in the characterization

of the pathogen of leaf blight and crown rot.

MATERIALS AND METHODS

PART A: Fungus Characterization and Identification

Fungal isolate sources. The fungus, designated DCL, was isolated

in 1978 from diseased 'Toronto' creeping bentgrass plants from putting

greens in Chesterland, Ohio. Diseased plants were washed for ten minutes

in distilled water with two drops of Tween 20 (Emulsion Engineering,

Inc., Elk Grove, IL 60007) and then surface disinfested by soaking for

two minutes in 0.5% sodium hypochlorite. Plants were then aseptically

plated on lactose casein hydrolysate medium (LCH) (Malca and Ullstrup,

1962) and incubated at 20C under fluorescent light at 5.5 Klux.

Mycelia and conidiophores could be observed first on host tissue and

later on the medium surface. Mycelia and conidia of this isolate (DCL)

were transferred into pure culture and maintained on LCH. All isolations

made throughout this research were done in this manner, regardless of

whether leaf or stolon tissue was used.

Monoconidial isolations of DCL were made after four days by

removing individual conidia from the plated 'Toronto' bentgrass tissue

using a very thin glass fiber. The glass fiber was made by heating and

then drawing out the narrow end of a disposable glass pipette into a

fine thread. Single conidia were placed on the surface of two percent

water agar (Difco Laboratories, Detroit, MI 48201) plates (WA) and

observed for germination after 24 hours at 20C. Plugs of agar

12

13

containing monoconidial colonies of the fungus were transferred to

separate LCH plates. Monoconidial isolates, designated DCL-2 and

DCL-4, were used in this study.

Other isolates used in this study included: a) Q. "catenaria (DCZ)

from reed canarygrass (fhalaris aruudinacea), courtesy of K. E. Zeiders,

u.s. Regional Pasture Lab., Agricultural Research Service, U.S. Depart-

ment of Agriculture, University Park, PA 16802; b) Q. erythrospila (DE)

from Agrostis palustris (isolate #291, collected in Cuyahoga Co., OH);

c) Q. dictyoides f. sp. dictyoides (DDD-O) from Festuca arundinacea

(isolate #1076, collected in Franklin Co., OH); d) Q. dictyoides f. sp.

dictyoides (DDD) from Festuca arundinacea (isolate #EC-3 L-l 55-3,

collected in Pennsylvania); and, e) D. dictyoides f. sp. perenne (DDP)

from Lolium perenne (isolate #HD WD LD SS-6, collected in New Jersey).

The isolates of DDD and DDP are courtesy of R. H. Morrison, Northrup King

Co., Eden Prairie, MN 55344. Stock cultures of the isolates were stored

on silica gel (Sleesman et aI, 1974).

Herbarium specimen sources. Type collection specimens of

Helminthosporium catenarium and ~. dictyoides used for comparative

studies were on loan from the following institutions:

a) ~. catenarium TYPE (DCT) on Ciuna arundinacea collected atD~uglaston, New York, 26 September 1920, by C. Drechsler, onloan from The National Fungus Collections, AgriculturalResearch Center - West, Beltsville, MD 20705.

b) ~. dictyoides (DDT) on "Festuca elatior collected at PortWashington, New York, July 1920, by C. Drechsler on loanfrom The Farlow Herbarium of Harvard University, 20 DivinityAvenue, Cambridge, MASS. 02138.

14

c) li. dictyoides TYPE (DDT) on Festuca elatior collected nearPort Washington, New York, 12 July 1920, by C. Drechsler,on loan from The New York Botanical Garden, Bronx, NY 10458.

·Conidia collection. The conidia and conidiophores of DCL, DCZ

and DDD were collected from the surfaces of host tissue plated on LCH.

Naturally infected 'Toronto' creeping bentgrass leaf tissue was the

source of DCL conidia and conidiophores,-and artificially inoculated

leaves were the sources of DCZ and DDD conidia.

The effect of media composition on conidial morphology was

studied using different culture media and three isolates of D. catenaria

(DCL, DCL-2 and DCL-4). Separate petri plates containing 15 ml of one

of three types of media were used. The media types included: potato-

dextrose agar (Difco Laboratories, Detroit, MI 48201) (PDA), V-8 juice

agar (Diener, 1955) (V-8JA) and LCH medium, all adjusted to pH 6 with

sodium hydroxide or hydrochloric acid. Conidia were collected from

respective cultures one cm from the margin of the expanding colony

seven days after incubation at 20C with a 12 hr day of 5.5 Klux.

Microscopic measurement of conidial characters. For each species

or isolate, whether from actively growing cultures or dried specimens,

25 representative conidia were observed. Various morphological

characters were measured or noted, including ones suggested by R. A.

Shoemaker (1962). Only mature, four or more celled conidia were

measured in all tests. Conidia were mounted on glass slides in lacto-

phenol (85% lactic acid, glycerine, phenol and water (1:1:0.5 v/v)) and

measured at 100X magnification using an ocular 5 rom microscopic micro-

meter disc (American Optical Corp., Buffalo, N.Y. 14215).

15

The conidial measurements taken included: total length (L),

width (W), length basal cell (BC) , width basal septum (BS), width apical

septum (AS) and distance from the widest part of the conidium to the

conidium base (PWP). Descriptive data included: number of septa,

widest cell, basal cell shape, color and scar appearance. Measurements

are reported as means and ranges with sum totals of measurements used

in calculating certain ratios: the length to width ratio (L/W),

the ratio of width of apical septum to basal septum (AS/BS), the ratio

of the distance of the widest part from the conidium base to conidium

length (WP), and the ratio of conidium length to number of cells,

referred to as the average cell length (ACL). Dimensional data and

descriptive information about conidiophores were only taken on DCL and

included: total length, length of each cell, width, and the number

of cells.

Origin and maturation of conidia. An agar block technique was used

to observe conidiogenesis and maturation of conidia of the bentgrass

isolate. Water agar (2%) blocks 1 x 1 x O.4cm thick were placed in

the center of separate sterile glass slides. The sides of the blocks

were seeded with DCL mycelia and then the blocks were covered with a

22 x 22 mm coverglass. The slides were incubated in moisture chambers

at 20C with fluorescent lighting (5.5 K lux) for 12 hr daily. Moisture

chambers were created by half-filling a covered glass petri dish with

sterile distilled water and supporting the slides above the water on

bent glass rods. After four days, the slides were removed from the

moisture chambers and observed at lOOX during a several hour period.

16

Sclerotium-like body formation. Discs of a ten-day old LCH culture

of isolate DCL were placed in the center of autoclaved 'Toronto'

bentgrass leaf clippings that had been aseptically placed on the surface

of water agar plates. Discs of the fungus were also placed on the

surface of bentgrass extract agar (BEA) prepared by adding 20g water

agar to one liter of the extract water from the autoclaved leaf clippings.

The seeded BEA arid leaf clipping plates were incubated at 20C with

fluorescent lighting (5.5 K lux) for 12 hr daily. The presence of

sclerotium-like bodies on tissue and/or in the medium was recorded

after 14 days.

Conidium germination. The mode of conidial germination was

observed on conidia mounted in sterile distilled water on glass slides

incubated at 2lC exposed to fluorescent lighting. A conidium was

considered germinated when the germ tube was twice as long as it was

wide.

Differential pigment production in culture. Differential pigment

production by Drechslera isolates was determined as described by

Shoemaker (1962). Discs of mycelia from ten-day old LCH cultures of

DCL, DCL-2, DCL-4, DCZ, DDD, DDD-O, DDP and DE were transferred to

polystyrene disposable petri dishes containing 15 ml of sucrose proline

agar (SPA), and incubated at 20C in the dark. After seven days, the

diffusion of pigment into the agar away from the colony, was recorded

for each of five replications per isolate. Color intensity was recorded

as slight, moderate or dense.

17PART B: Host Range Study

Host species. Six bentgrasses, four Kentucky bluegrasses, two

fescues and two other forage grasses were used in the inoculation

studies. The bentgrasses included one colonial bentgrass (Agrostis

tenuis cv. Highland) and five creeping bentgrasses(~. palustris

cultivars Emerald, Cohansey, Penncross, Seaside, Toronto). The

Kentucky bluegrasses (Poa pratensis) included the cultivars Adelphi,

Delta, Merion and Park. Other grasses used in the study were tall

fescue (Festuca arundinacea cv. K-3l), red fescue (F. rubra cv. Pennlawn),

perennial ryegrass (Lolium perenne cv. Manhattan) and reed canarygrass

(Phalaris arundinacea).

Plant cultivation. All grasses were established from seed except

'Cohansey' and 'Toronto' creeping bentgrass. These cultivars were

established from sod obtained from Warren Turf Nurseries (8400 West

lllth Street, Palos Hills, IL 60464). No plant pathogens were isolated

from this vegetative material. The bentgrasses (0.3 g seed/pot) as

well as the other grasses (0.5 g seed/pot) were seeded in 8.8 cm

styrofoam cups filled with sterile potting soil mixture consisting of

soil, peat and perlite (1:1:1 v/v). The grasses established from sod

were also cultivated in this soil mixture. The pots of plants were

maintained in the greenhouse, and clipped weekly to a height of 2-3 cm

for the bentgrasses and 5-10 cm for the other grasses. The plants

were watered daily and treated with 200 mg/L of a 15-15-15 water soluble

fertilizer. The plants were three to five months old when used in

experiments.

18

Inoculum preparation. Isolates of the actively growing cultures

were grown for 14 days on LCR at 20C with 12 hr of fluorescent and

incandescent light (5.5 K lux) daily. Conidia of each isolate were

harvested by flooding each plate with 10 ml of a sterile distilled

water - Tween 20 solution (2 drops Tween 20 per 100 ml water) and gently

rubbing the surface of the colony with a bent glass rod to dislodge the

conidia. The conidial suspension was strained through a single layer

of 80 mesh cheesecloth and diluted to ca. 10,000 conidia/ml of distilled

water. A Sedgwick-Rafter counting cell (VWR Scientific, Columbus, OR

43215) was used in conjunction with an ocular microscopic Howard disc

(American Optical Corp., Buffalo, NY 14215) to standardize the conidial

concentration.

Inoculation procedure. In experiments to determine the patho-

genicity of Drechslera isolates, 1.0 ml of the appropriate conidial

suspension was uniformly applied to the foliage of the respective grass

species with an artist's air brush (Badger Air Brush Co., Franklin

2Park, IL 60131) at 0.7 kg/cm. The check plants were sprayed with

water plus Tween 20 only. Pots of inoculated plants were sealed in

polyethylene bags and incubated in growth chambers for 60 hours at 25C

with fluorescent and incandescent lighting for 12 hr daily. The plants

were maintained at 25C throughout the experiment and watered and

fertilized daily. Each experiment was replicated three times with

three subsamples per replication. A subsample consisted of a single

pot, resulting in a total of nine subsamples for each treatment of the

experiment.

19

Disease severity. Incidence and severity of the disease symptoms

were evaluated two weeks after inoculation. The pots were rated

according to the percent area of the pot that had diseased plants

using a rating scale where: 1 no disease, 2 = 1-5%, 3 = 6-25%,

4 = 26-50%, 5 = 51-75% and 6 76-100%. Pathogens were isolated on

LCH medium as previously described or by incubation on moist filter

paper for seven days. There were no symptoms on any of the check

plants. The disease severity ratings were analyzed according to the

Duncan's New Multiple Range Test at the five percent level of

significance.

RESULTS

PART A: Fungus Characterization and Identification

The pathogen. The conidia of the Drechslera catenaria isolate

from Agrostis palustris 'Toronto' (Fig. 1.1) are hyaline to light

yellow with a thin wall and dark hilum. Conidia are obclavate (Fig. 1.2),

tapering to a narrow apex (AS/BS 0.50 - 0.71, avo 0.62), from the

widest point (WP) at 0.15 - 0.25 (av. 0.17) which corresponds to the

first septum or second cell from the base. Basal cells are 15 - 20.7 ~m

(av. l8.4~m) long and basal septa are 13.8 - 19.6~m (av. l6.l~m) wide,

giving the basal cells a hemi-ellipsoidal shape. Basal scars are

within the contour of these cells. Conidia measure 73.6 - 126.5 x

13.8 - 19.6~m (av. 110.0 x l6.2~m), usually with 3-6 septa (av. 4.9),

L/W 4.4 - 8.8 (av. 6.8) and average cell length of l8.7~m.

Conidiophores (Figs. 1.1 and 1.3) are medium yellowish brown,

simple, arising singly or in small groups especially from leaf veinal

regions, with the conidia borne singly at the tips. Conidia are

catenate (Fig. 1.4), in chains of 2-4 or more with secondary

conidiophores present between conidia, leaving an apical conidial scar

at the point of attachment. Primary conidiophores are 57.5 - 94.3~m

(av. 76.3~m) long and 2-3 septate with short enlarged foot cells

(6.9-20.7, avo 11.2~m), with the middle (13.8-39.1, avo 26.8~m) and

the apical conidiophore cells (23.0-50.6, avo 38.3~m) being much longer.

20

Figure 1.1 Conidia and conidiophores of Drechs1era catenaria onincubated 'Teronto' creeping bentgrass leaf tissue(100X).

21

Figure 1.2 Conidia from culture of Drechslera catenaria grown onlactose casein hydrolysate medium (400X).

22

Figure 1.3 Drawings of conidia and conidiophoresof Drechslera catenaria from incubated'Toronto' creeping bentgrass leaftissue. A-B) Germinating conidia.C) Secondary conidia with adjoiningconidiophore. D-E) Primaryconidiophores on leaf tissue.

23

24

Figure 1.4 Primary and secondary conidia and conidiophores ofDrechslera catenaria. A) from leaf tissue, (80X).B) from culture on lactose casein hydrolysatemedium (200X). Photo B courtesy of A. K. Hagan.

25

A

26

27

The conidiophore stalks are uniform in diameter (5.6-8.I~m). The

apical tips are slightly swollen and bear scars.

Spore germination in distilled water occurred after one to two

hours. Germination occurred from end cells, one or more of the inter­

calary cells or a combination of both. Germ tubes radiate laterally

or semiaxially from the basal and intercalary cells, but emerge

terminally and radiate with the long axis of the spore from the apical

cells. More than one germ tube may emerge from each cell, especially

the basal cell, but germ tubes do not emerge through the hilum.

Numerous dark globose sclerotia-like bodies (SLB) were found

embedded in both the autoclaved leaf tissue and in the bentgrass

extract agar medium after two weeks. The SLB formed from thickened

mycelia and were found at or near the bottom of the extract plates.

The conidia appeared to have originated as a protrusion through

a pore in the conidiophore apex wall (Fig. 1.5a). The immature conidium

then expanded to a cylindrical form (Fig. 1.5b). The first septum

delimited the basal cell (Fig. 1.5c); the conidium continued to elongate

(Figs.l.5d and 1.5e); the next septum formed near the middle of the

cell (Fig. 1.5f) and additional septa formed in the apical and basal

regions (Figs. 1.5g and 1.5h). This conidiogenic sequence occurred

within four hours.

The colony characteristics of DCL differed according to the culture

medium composition (Fig. 1.6). Moderately erumpent colonies were

produced on lactose casein hydrolysate medium that were initially white

to tan, later turning olive-gray. On potato dextrose agar, DCL produced

28

b8·

Figure 1.5 Origin and maturation stages of a conidium ofDrechslera catenaria, (200X). a-b) Origin ofa conidium. c-h) Stages in septation (arrows)of a conidium.

A B

29

Figure 1.6 Colonies of Drechslera catenatia on three culture media.Ten-day-old colonies on A) V-8 juice agar, B) lactosecasein hydrolysate medium, C) potato-dextrose agar afterincubation at 20C and 12 hr daily illumination (5.5 Klux).

30dense erumpent colonies, slate gray to bla~k. Colonies were pale

gray and only moderately erumpent on the V-8 juice agar. Concentric

bands of white aerial hyphae on the V-8JA colonies corresponded

approximately with the dark period. Sporulation occurred on all three

media with 12 hr daily illumination. The fungus did not sporulate

when incubated in constant darkness. No SLB were observed in any of

the cultures.

Comparison ~ conidial dimensions of the pathogen with~

collection specimens and known isolates. Conidial dimensions and

morphology of D. catenaria isolates DCL and DCZ and D. dictyoides

isolate DDD were compared to those of the type collection specimens of

Drechslera catenaria (DCT) and Q. dictyoides (DDT) (Table 1.1).

Both Q. catenaria and D. dictyoides have spore dimensions that

are quite variable. In all categories of observation, the ranges

overlapped among all isolates. However, there were some significant

differences in measurements between the two type specimens. The

conidia and basal cells of the D. catenaria type specimen were longer

(89.5, 18.l~m) and the conidia were wider (16.4~m) than those of

Q. dictyoides (69.2, 13.6 and 14.8~m, respectively). The width of the

apical septum was narrower for DCT (9.0~m) than for DDT (10.7~m),

but the width of the basal septum and the number of cross septa per

conidium did not differ significantly. The conidia of DCT also tapered

more sharply from the base to the apex, that is having a lower AS/BS

ratio (0.67 to 0.80), and had longer average cell lengths (16.7~m) than

did the DDT conidia (13.7~m).

Table 1.1.. Comparison of the morphological characters of conidia from Drechs1era tatenaria frombentgrass (DCL) with type specimens and known isolates of D. catenatia and Q. dictyoides. w

Character DCTx

DCL DCZ DDT DDD

Lengths lJm89.5BYConidium (L) 110.0A 110.7A 69.2C 110.7AB

z73.6 - 126.5 78.2 - 142.6 48.3 - 87.4 78.2 - 117.357.5 - 128.8

Basal Cell 18.lA 18.4A 16.0B 13.6C 16.4B11.5 - 25.3 15 - 20.7 11.5 - 18.4 11.5 - 16.1 13.8 - 20.7

Widths lJmConidium (W) 16.4A 16.2A 17.0A 14.8B 17.6A

10.4 - 23.0 13.8 - 19.6 13.8 - 20.7 11.5 - 16.1 13.8 - 25.3Apical Septum (AS) 9.0D 10.0BC 9.7CD 10.7AB 11.lA

4.6 - 13.8 9.2 - 13.8 6.9 - 11.5 8.1 - 13.8 9.2 - 13.8Basal Septum (BS) 13.6B 16.lA 15.8A 13.4B 15.6A

9.2 - 18.4 13.8 - 19.6 12.7 - 18.4 10.4 - 16.4 11.5 - 20.7

Septa (No.) 4.5BC 4.9AB 5.0AB 4.1C 5.2A3 - 7 3 - 6 4 - 7 3 - 6 4 - 8

Average Cell 16.7B 18.7A 18.5A 13.7C 16.6Blength (ACL) lJm 10.7 - 23.0 13.3 - 23.5 13 - 23.9 10.6 - 16.7 13 - 20.2

AS/BS Ratio 0.67BC 0.62C 0.61C 0.80A 0.72B0.43 - .9 .5 - .71 0.38- .83 0.67- 1.00 0.5 - .92

L/W ratio 5.7B 6.8A 6.6A 4.7C 5.8B3.7 - 9.0 4.4 - 8.8 4.1 - 10.0 3 - 5.8 4.2 - 7.5

Wide point (WP) 0.28A 0.17C 0.19C 0.19C 0.23B0.16 - .41 0.12- .25 0.1 - .36 0.13- .26 0.14 .32 w.....

Table 1.1. (Continued)

w

x

y

z

Conidia of DCT and DDT from herbaria collections; Conidia of DCL, DCZ and DDD produced onincubated live or dead leaf tissue at 20C with a daily 12 hr exposure to cool-white fluorescentlight (5.5 K lux) collected after 10 days.

Isolates: DCT = Drechslera catenaria (TYPE), DCL = Q. catenaria (from Agrostis palustris),DCZ =~. catenaria (from Phalaris arundinacea), DDT = D. dictyoides (TYPE) and DDD = Q. dictyoidesf. sp. dictyoides (from Festuca arundinacea).

Means are averages of measurements of 25 representative conidia. Means followed by the sameletter in rows are not significantly different (P = .05) according to the Duncan's New MultipleRange Test.

Ranges listed below means are extremes of conidia observed.

WN

33

The dimensions of the conidia from the active cultures were

different from those recorded for the type specimens of their respective

species. The Q.dictyoides isolate DDD had many conidial dimensions

that were intermediate between the measurements of the D~catertaria

isolates (DCL, DCZ) and the Q~ dictyoides type specimen (DDT). The

Q. catenatia isolates matched more closely in conidial dimensions with

the type, but did exceed the D~ catertatia type specimen (DCT) measurements,

especially in length, average cell length and septum width. The widest

point of the conidium was also further from the base, indicated by a

larger WP, for DCT than with DCL or DCZ.

Some of these conidial characters appear to be more important than

others in the separation and identification of Drechslera species

(Shoemaker, 1962). Those especially useful are conidium length (L),

length basal cell (BC), average cell length (ACL) and the AS/BS ratio.

A visual comparison of these characters is presented in Fig. 1.7. The

vertical lines indicate the range of conidial measurements and the

horizontal lines are the averages.

Differential pigment production. Actively growing Drechslera

colonies on sucrose proline agar produced pigments that diffused into

the agar (Table 1.2). All D. catenaria isolates (DCL, DCL-2, DCL-4 and

DCZ) produced a yellow or yellow-orange pigment that was moderate to

dense in intensity (Fig. 1.8). Dtechsleradictyoides f. sp. dictyoides

(DDD gnd DDD-O) and Q. dictyoides f. sp. perertne produced a pink pigment

that varied in intensity. Q.etythrospila produced a brown-green

pigment with limited diffusion away from the margin of the colony.

34

Figure 1.7 Comparison of conidial length, length basal cell, averagecell length and AS/BS ratio of conidia of Drechsleracatenaria and Q. dictyoides isolates. DCL = D. catenariafrom creeping bentgrass, DCZ = Q. catenaria from reedcanarygrass, DCT = D. catenaria from wood reedgrass(Type), DDT = Q. di~tyoides from meadow fescue (Type),DDD = Q. dictyoides from tall fescue. Vertical barsindicate ranges and horizontal bars indicate meanvalues from 25 randomly selected conidia.

35

DRECHSlERA CATENARIA ON D. DICTYOIDES ON

BENTGRASS(DCl)

REEDCANARYGRASS(DCZ)

WOODREEDGRASS(OCT)

MEADOWFESCUE(DOT)

TAllFESCUE(000)

E::t 120

J: t....~IOOw...J

;i 80 taz8 60

40

E..::- 24...J...Jw

t tu 20...J

t<t(/)

<t 16

+CD

J:....C)

12zw...J

J:....C)zw 20...J...J...J-wEu::lJ6wC)<ta: 12w><t

(/)

t t tCD....... t(/)

<t .6J

.4

Table 1.2. Differential pigment production of Drechslera spp. inculturea

36

Drechslera Pigment Color bspecies Isolate .Host. colorb Intensity

Q.. catenaria DCL Agrostis palustriscv. Toronto Yellow 3

D. catenaria DCL-2 A. paltistiiscv. Toronto Yellow 2

D. catenaria DCL-4 A. palustriscv. Toronto Yellow 3

D. catenaria DCZ Phalarisarundinacea Yellow-orange 3

D. dictyoides Festucaf.sp. dictyoides DDD-O arundinacea Pink 2

D. dictyoidesf.sp. dictyoides DDD F. arundinacea Pink 3

D. dictyoidesf.sp. perenne DDP Lolitnn perenne Pink 1

D. erythrospila DE A. palustris Brown-green 2

a

b

Pigment production in agar observed after seven days whencultured on sucrose proline agar at 20C in the dark(Shoemaker, 1962).

Pigment color and intensity of color judged as overallimpression of five plates of each isolate. Intensity ofcolor judged where: 1 slight pigmentation, 2 = moderatelydense pigmentation and 3 = very dense pigmentation.

37

Figure 1.8 Pigment production by Drechslera catenaria and Q. dictyoidesisolates on sucrose proline agar after ten days at 20C.DCL = Q. catenatia from 'Toronto' creeping bentgrass,DCZ = Q. catenatia from reed canarygrass, DDD =D. dictyoides from tall fescue.

38

All colonies were white to pale gray with moderate growth and no evident

sporulation or protothecia production under these conditions.

Factors affecting conidial characters of Drechslera catenaria

isolates in culture. When the isolates of Drechslera catertaria

(DCL, DCL-2 and DCL-4) were compared on three culture media, differences

in conidial morphology were more evident between media treatments than

between isolates (Table 1.3). Conidial length, length of basal cell,

width of basal septum, number of septa, L/W ratio and AS/BS ratio did

not differ significantly between isolates on any medium. Width differed

significantly, with DCL-4 commonly widest on LCH and PDA, but DCL-2 was

widest on V-8 JA. Apical septum width varied among isolates on all

three media, again with the septa of conidia of DCL-2 and DCL-4

significantly wider than those of DCL. DCL had longer average cell

lengths on LCH.

There were significant effects of the culture medium on conidial

dimensions when averaged over the isolates (Table 1.4). There were

highly significant differences in length, width, L/W ratio, average cell

length and basal cell length of the conidia from the three growth

media and also in number of septa. Measurements of conidia produced

on LCH were significantly larger than those observed from the other

media. The widths of the apical and basal septa were significantly

greater on LCH and PDA than on V-8JA. The AS/BS ratio was the only

value unaffected by culture medium.

Effect of culture substrate on the variation of conidial characters

of the~ isolate of D. catenaria. The characters of conidia produced

Table 1.3. The effect of culture medium on the conidial characters of three Drechslera catenariaisolates from Agrostis palustris cv. Toronto. w

Character C id'Basal Apical Average

on ltml Basal Cell Coniditml Septum Septum Septa CellMediumx IsolateY Length(vm) Length (llm) Width (llm) Width(pm) Width (llm) (no.) Length(pm) AS/BS L/t~

LCH DCL 131.4 AZ 19.0 B 16.8 C 15.7 AB 9.8 BCD 5.2 BC 22.1 A 0.63 A 7.9 A

DCL-2 129.2 A 19.6 AB 17.4 B 16.2 A 10.5 AB 5.6 AB 19.4 B 0.65 A 7.5 A

DCL-4 134.6 A 20.7 A 17.9 A 16.7 A 10.7 A 5.8 A 20.1 B 0.64 A 7.7 A

PDA DCL 98.8 B 17.0 CD 16.3 E 16.0 AB 9.6 CD 4.7 CDE 17.6 C 0.61 A 6.1 B

DCL-2 93.4 BC 18.3 BC 16.6 D 16.4 A 10.7 A 5.0 CD 15.6 D 0.66 A 5.7 BC

DCL-4 100.3 B 19.6 AB 16.8 C 16.2 A 10.2 ABC 5.0 CD 17.0 C 0.63 A 6.0 BC

V-BJA DCL 88.0 C 16.6 D 14.8 G 14.1 C 9.2 D 4.6 DE 16.0 D 0.66 A 6.0 BC

DCL-2 84.7 C 17.3 CD 16.2 E 15.1 B 10.0 ABCD 4.4 E 15.9 D 0.66 A 5.3 C

DCL-4 84.6 C 17.3 CD 15.4 F 15.0 BC 10.1 ABC 4.5 DE 15.5 D 0.68 A 5.6 BC

w Conidia produced at 20C with daily 12 hr exposure to cool-white fluorescent light (5.5 K lux) for10 days.

x Media: LCH = Lactose casein hydrolysate medium, PDA = potato' dextrose agar medium and V-8JA = 20%V-8 juice medium.

y Isolates: DCL = mass isolate, DCL-2 and DCL-4 = monoconidial isolates; all three fromAgrostis palustris 'Toronto'.

Z Means are averages of measurements of 25 representative conidia. Means followed by the same letter in wcolumns are not significantly different (P = .05) according to the Duncan's New Multiple Range ~

Test.

40

Table 1.4. The effect of culture medium on the conidial charactersof Drechslera catertaria averaged over isolates. x

MediumY

Character . 'LCH PDA V-8JA

Lengths (lIm)A

ZConidium (L) 131.7 97.5 B 85.8 C

Basal cell (BC) 19.7 A 18.3 B 17.1 C

Widths (lJm)Conidium (W) 17.4 A 16.6 B 15.5 C

Apical septum (AS) 16.2 A 16.2 A 14.8 B

Basal septum (BS) 10.3 A 10.2 A 10.0 B

Septa (No.) 5.5 A 4.9 B 4.5 C

Average celllength (ACL) (lJm) 20.5 A 16.7 B 15.8 C

AS/BS ratio 0.64 A 0.63 A 0.67 A

L/W Ratio 7.7 A 5.9 B 5.6 B

x

Y

Z

Conidia produced at 20C with daily 12 hr exposure to cool-whitefluorescent light (5.5 K lux) for 10 days.

Media: LCH lactose casein hydrolsate medium, PDA = potato~

dextrose agar medium and V-8JA = 20% V-8 juice medium.

Means averaged over three isolates (DCL, DCL-2 and DCL-4) foreach medium. Means are based on measurements of 75 representativeconidia, 25 from each isolate. Means followed by the same letterin rows are not significantly different (P = .05) according tothe Duncan's New Multiple Range Test.

41

on host tissue were compared with those of conidia produced on three

culture media using only the DCL isolate (Table 1.5). All substrates,

~ncluding host tissue, affected conidial length. Conidia from LCH were

longest (13l.4~m) and conidia from V-8JA were shortest (88.0~m).

Conidia produced on host tissue had a shorter ACL and a smaller L/W

ratio than those on LCH but there was no difference in basal cell

length, conidial width or basal septum width. Values from conidia

produced on V-8JA were lowest in every character category.

Variation in conidial characters due to substrate was compared by

calculating the coefficient of variation (Little and Hills, 1978) and

is presented along with the data in Table 1.5. Conidial length,

number of septa, ACL and L/W ratio all varied nearly 20% when the

population of conidia was observed, regardless of substrate. However,

the BC, W, AS, BS and AS/BS ratio values only varied 10-15%. The

variation in conidial measurements within each population of 25 conidia,

as affected by substrate, is not presented. Only the composite C.V.

over characters is shown for each substrate. Variation of all conidial

characters was least for those produced on host tissue with a

coefficient of variation of only 12.3%. Variation was least for those

produced on host tissue in seven of the nine character categories with

all values less than 16%. The variation of the AS/BS ratio and AS

was lowest on LCH.

Table 1.5. The effect of culture substrate on the conidial characters of Drechs1era catenariafrom Agrostis pa1ustris cv. Toronto. w

SubstrateXC. V. (%)Y

OverCharacter Leaf Tissue LGH PDA V-8JA Substrate

Lengths llm110.0 BZConidium (L) 131.4 A 98.8 C 88.0 D 21.0

Basal cell (BC) 18.4 A 19.0 A 17.0 B 16.6 B 12.8

Widths llmConidium (W) 16.2 A 16.8 A 16.8 A 14.8 B 12.4

Apical septum (AS) 10.0 A 9.8 A 9.6 A 9.2 A 13.6

Basal septum (BS) 16.1 A 15.7 A 16.0 A 14.1 B 11.8

Septa (no.) 4.9 A 5.2 A 4.7 A 4.6 A 20.2

Average celllength (ACL) llm 18.7 B 22.1 A 17.6 B 16.0 C 19.3

AS/BS ratio 0.63 A 0.63 A 0.61 A 0.66 A 14.7

L/W ratio 6.8 B 7.9 A 6.1 C 6.0 C 20.7

C. V. (%) over 12.3 15.6 14.8 13.7charactersY

.t:'N

Table 1.5. (Continued)

w

x

y

z

Conidia of Q. catenaria isolate (DCL) produced at 20C with a daily 12 hr exposure tocool-white fluorescent light (5.5 K lux) collected after 10 days.

Substrates: Tissue = Conidia produced on Agrostis palustris diseased tissue, LCHlactose casein hydrolysate medium, PDA = potato-dextrose agar and V-8JA = 20% V-8juice agar.

Coefficient of variation (CV) expressed as a percentage and calculated over substratesfor each conidial character and calculated over all characters for each substrate.

Means are averages of measurements of 25 representative conidia. Means followedby the same letter in rows are not significantly different (P = 0.05) accordingto the Duncan's New Multiple Range Test.

.p.VJ

44

PART B: Host range study

Host range. The pathogenicity of the fungus isolated from

'Toronto' bentgrass (PCL) has been proven (Larsen et aI, 1980) but

the host range reported was limited. Results of a more extensive host

range study using isolate DCL conducted by artificially inoculating

greenhouse-grown turf and forage grasses are given in Table 1.6. All

creeping and colonial bentgrass cultivars inoculated were susceptible

to this isolate. Disease severity was significantly higher for the

bentgrasses than most other grasses with cultivars Toronto (5.0),

Emerald (3.4), Highland (3.2) and Penncross (3.1) rating the highest

with bentgrass cultivars Seaside (2.4) and Cohansey (2.4) less

severely affected. The pathogen was isolated from 95-100% of diseased

leaves sampled. Sporulation of the fungus was observed only on incubated

necrotic tissue.

The typical disease symptoms on bentgrass began as small reddish­

tan lesions or bands on the leaf blades (Fig. 1.9). After several

weeks, there was tip dieback and extensive blighting of entire blades.

These symptoms were similar to those observed on diseased 'Toronto'

bentgrass putting greens. Reed canarygrass (Phalaris arundinacea)

was also affected by this pathogen (2.7) which resulted in a leaf tip

dieback.

Inoculation of tall fescue (K-3l), 'Manhattan' perennial ryegrass

and 'Pennlawn' red fescue with Dr,L· ~solate resulted in the production

of very small (less than 1 rom diameter) reddish-brown necrotic local

lesions. This "flecking" of the leaf blades was reflected in the low

45

Table 1.6. Pathogenicity of Drechslera'catenaria (DCL) on variousgreenhouse grown turfgrass species and cultivars. x

HOST Disease SeverityY,

Agrostispalustris 'Toronto' 5.0 AZ

A. palustris 'Emerald' 3.4 B

A. tenuis 'Highland' 3.2 BC

A. palustris 'Penncross' 3.1 BCD

Phalaris arundinacea 2.7 CDE

A. palustris 'Seaside' 2.4 DE

A. palustris 'Cohansey' 2.4 DE

Festuca arundinacea 'K-3l' 2.0 EF

Lolium perenne 'Manhattan' 2.0 EF

F. rubra 'Pennlawn' 1.7 FG

Poa pratensis 'Adelphi' 1.0 G

P. pratensis 'Delta' 1.0 G

P• .EEatensis 'Merion' 1.0 G

P. pratensis 'Park' 1.0 G

x

y

Z

Pots of grass were inoculated with 10,000 conidia, incubated for60 hours under high humidity and maintained at 25C with 12 hoursof illumination per day throughout the experiment.

Disease severity was rated two weeks after inoculation using arating scale where 1 = no disease and 6 = 76-100% of the pot areadiseased.

Means followed by the same letter not significantly different(P = 0.05) according to Duncan's New Multiple Range Test. Meansare averages of three 'replication of three subsamples each.

Figure 1.9 Lesions on 'Toronto' creeping bentgrass caused byDtechs1era catenaria five days after artificialinoculation.

46

47

disease ratings (2.0, 2.0, 1.7) of 'K-3l', 'Manhattan' and 'Pennlawn',

respectively. Lesions did not expand or coalesce with time and were

commonly without a chlorotic halo. Lesions occurred mostly over veins,

but also interveinally. The fungus was reisolated from less than half

of the diseased leaves sampled and sporulation was very sparse. No

symptoms were observed on Kentucky bluegrass cultivars Adelphi, Delta,

Merion or Park, nor could the pathogen be reisolated. No disease

symptoms were observed on any uninoculated control plants in this study.

These grasses were also inoculated with the monoconidial isolates

(DCL-2 and DCL-4), and the pathogenicity results were similar to those

reported for the DCL isolate. All isolates of Q. catenaria (DCL, DCZ

and DCL-4) produced symptoms similar to those previously reported.

Comparative pathogenicity. The pathogenicity of Drechslera

catenaria (DCL), D. dictyoides f. sp. dictyoides (DDD), D. dictyoides

f. sp. perenne (DDP) and Q. erythrospila (DE) on ten turf and forage

grasses is presented in Table 1.7. The DCL results from this comparative

study and the previous study are similar.

DCZ, DDD and DDP were only slightly pathogenic on the bentgrasses,

but DE severely damaged all bentgrasses tested. Symptoms caused by

DE on 'Seaside' were more severe (4.2) than those caused by DCL

(2.0). However, DCL caused more severe symptoms on 'Toronto' (5.5)

than did DE (3.8). DCZ, DDD and DDP were most severe on the hosts

from which the pathogen was originally isolated (Phalaris arundinacea,

Festuca atundinacea andLoliumpetenne, respectively). The D. catenaria

isolate from reed canarygrass(~~atundinacea)caused only minor damage

Table 1.7. Pathogenicity of Drechs1era catenaria, Q. dictyoides f. sp. dictyoides, D. dictyoidesf. sp. perenne and D. erythrospi1a of various turf and forage grasses. w

DCLYDisease SeverityX

Host DCZ DDD DDP DE

Agrostis tenuis 'Highland' z1.3 b/c 1.8 b/bc 1.2 bide 4.0 ala3.3 alb

Agrostis pa1usttis 'Penncross' 3.2 alb 1.0 c/c 1.7 b/bcd 1.5 bc/cd 3.2 alb

A. pa1ustris 'Seaside' 2.0 bid 1.3 c/c 1.2 c/e 1.0 c/e 4.2 ala

A. pa1ustris 'Toronto' 5.5 ala 1.0 d/c 1.3 d/cde 2.0 c/b 3.8 b/a

Festuca arundinacea 'K-31' 2.0 c/d 2.2 bc/b 3.2 ala 2.2 bc/b 2.7 ab/b

F. rubra 'Pennlawn' 1.5 abc/e 1.3 c/c 1.5 abc/cde 1.8 ab/bc 2.0 a/c

Lo1ium perenne 'Manahattan' 2.0 bid 2.3 bib 2.2 bib 3.8 ala 2.5 b/c

Pha1aris arundinacea 2.7 b/c 3.7 ala (ND) (ND) 2.7 bib

Poa praterisis 'Adelphi' 1.0 alf 1.0 a/c 1.0 ale 1.0 ale 1.0 aid

P. pratensis 'Merion' 1.0 alf 1.0 a/c 1.0 ale 1.0 ale 1.0 aid

~

00

Table 1.7. (Continued)

w

x

y

z

Pots of grass were inoculated with 10,000 conidia, incubated for 60 hours under high humidityand maintained at 25C with 12 hours of illumination per day throughout the experiment.

Disease severity was rated two weeks after inoculation using a rating scale where:1 = no disease and 6 = 76-100% of the pot area diseased.

DCL = Drechslera catenaria (from Agrostis palustris), DCZ = Q. catenaria (fromPhalaris arundinacea), DDD = Q. dictyoides f. sp. dictyoides, DDP = Q. dictyoidesf. sp. perenne, DE = Q. erythrospila.

Means compared between fungal isolates on one host (across a/ ) and means compared betweenhosts with one isolate (down fa) followed by the same letter are not significantlydifferent (P = 0.05) according to the Duncan's New Multiple Range Test. Means areaverages of three replications of three subsamples each replication.

~\.0

50

to the bentgrasses and the D. catenaria isolate from creeping bentgrass

caused only moderate symptoms on reed canarygrass. None were patho­

genic on bluegrasses. No data were available for DDD or DDP on reed

canarygrass. No disease was observed in any of the control pots.

The DCL isolate caused a tip dieback and blight of the bentgrass

leaf blades~that began as small lesions that appeared five days after

inoculation. When diseased leaves were incubated in high humidity,

D. catenaria was reisolated from leaves of all bentgrass cultivars.

The fungus sporulated extensively on the necrotic tissue. Sporulation

was sparse on the incubated ryegrass, red fescue and reed canarygrass

blades. The pathogen was not recovered from bluegrass.

DCZ on reed canaryg~ass caused tip dieback and marginal chlorosis

of leaves. Blighting was more severe on the cut ends of blades. Tip

blight and bleached lesions were evident on blade edges of tall fescue

and perennial ryegrass as a result of DCZ inoculation. Sporulation

was abundant on incubated reed canarygrass leaves and the pathogen

was readily recovered. Sporulation was sparse on necrotic tissue of

tall fescue.

Drechslera erythrospila caused severe blighting of the leaf blades

of all bentgrass cultivars. Symptoms began as tan lesions or bands

across the leaf blades resulting in extensive blighting. DE caused a

mild tip blight on red fescue and perennial ryegrass. Symptoms

produced by DE on tall fescue were bleached lesions with reddish

borders that later resulted in leaf tip blight. The fungus was isolated

51

from all affected grasses after four days. Sporulation was abundant

on necrotic tissue of 'Highland', 'Penncross', 'Toronto', tall fescue

and perennial ryegrass.

Both DDD and DDP caused minor tip blight of the bentgrasses,

but only a few leaves were affected. The most severely affected

by DDP was perennial ryegrass (3.8) which had extensive tip blighting,

the tissue turning dark as it became necrotic. DDP caused flecking

with or without chlorosis on red fescue and tall fescue. DDP was

recovered from red fescue, tall fescue and ryegrass with sparse to

moderate sporulation.

DDD most severely affected tall fescue (3.2). The tip blight of

this host began as expanding lesions which coalesced to cause

extensive leaf blight. DDD caused a slight tip blight on perennial

ryegrass. DDD was recovered from 'Emerald', 'Highland', 'Pennlawn' and

'K-3l'. There was sparse sporulation on incubated necrotic tissue

of the bentgrasses.but abundant sporulation on the necrotic fescue

tissue.

DISCUSSION

The conidia of the fungal isolate from creeping bentgrass (DCL)

are similar in morphology with conidia described by Drechsler for

Helminthosporium catenarium (1923). The long conidia are obclavate,

tapering to approximately half the maximum width at the tip. The

contour of the basal cell is hemi-ellipsoidal without a protuberant

hilum. Conidia of this isolate are catenate, forming chains with

short secondary conidiophores connecting adjacent conidia. Conidia

germinate amphigenuou3ly, with germ tubes arising indiscriminately

from any point on the conidium and from any cell. The germ tubes then

extend laterally away from the conidium. A developing conidium matures

acrosporously, whereby cells are delimited and mature in sequence

from base to apex as the tip of the conidium continues to expand.

In pure cultures on potato glucose agar, Drechsler reported that

H. catenarium produced a white to dirty yellowish aerial mycelium,

which is in contrast to that of DCL which was initially white but

later turning dark gray on potato-dextrose agar. The original

description of this fungus also did not report the production of

sclerotium-like bodies, as was found in this study on incubated host

tissue and in bentgrass extract agar.

In any taxonomic scheme, ranges in dimensions and variations

within a group commonly occur. As Shoemaker (1959) stated, "conidial

52

53

shape appears to be a useful character in the separation of species of

Drechslera t although differences can be expressed only as tendencies."

Frequent variation in conidial morpho~ogy in this genus has been reported t

with dimensions that exceed the original descriptions in some cases

(Kafi and Tarr, 1966; Tarr and Kafi, 1968). Other researchers have

indicated previous misidentifications of Drechslera species because

of this variation (Shoemaker, 1962; Morrison, 1980).

Within the genus Drechslera, only three species have conidia

with a pronounced taper towards the apex: Q. catenaria, ~. dictyoides

and D. phlei. Although conidia of D. phlei are tapered, there is a

constriction at the basal septum with the second cell from base

abruptly enlarged (Graham, 1955; Shoemaker, 1962). This character

prohibits the confusion with other Drechslera species. However,

D. catenaria and ~. dictyoides may not be as easily distinguishable

from one another (Labruyere t 1977; Luttrell, 1951; Scharif, 1961).

Differences between D. catenaria-type and Q. dictyoides-type

collection specimens t previously reported in the literature, were

confirmed by observation of these type specimens and respective active

cultures. Variation in dimensions occurred frequently but certain

"tendencies", some significant and others not, could be observed.

However, in all cases conidial dimensions of the active cultures

exceeded measurements of the conidia from the type collection

specimens. The conidia and basal cells of the Q. catenaria type

specimen (DCT) were longer and wider than those of the Q.dictyoides

type specimen (DDT). The conidia of DCT were also more tapered and had

54

longer average cell lengths. Conidia of the D. dictyoides type had

basal cells that were herni-spherical and not herni-ellipsoidal as in

Q.catenaria, as reported previously (Drechsler, 1923). Slight

constriction of the basal septum of Q.dictyoides was observed but

not as striking as indicated by Chidambaram et al (1973) and Morrison

(1980).

55

structures in their cultures of D. catenaria, but Zeiders (1976) did.

The Q. dictyoides isolate from Festuca sp. and Q. erythrospila both

produced sclerotium-like bodies on LCH, but D. dictyoides from perennial

ryegrass did not. These results were similar to those previously

reported for these fungi on other media (Braverman and Graham, 1960;

Luttrell, 1951; Shoemaker, 1962).

Successful inoculation of creeping and colonial bentgrass cultivars

with a Drechslera catenaria isolate had not been demonstrated prior to

the report by Larsen et al (1980). Artificial inoculations with

D. catenaria from C. arundinacea by Braverman and Graham (1960) and

Graham (1955) did not result in disease of several grass species. It

is evident from the damage observed on golf course greens and severe

damage on inoculated cultivars of bentgrass, that DeL is a destructive

pathogen and a potential threat to all midwest golf courses,

expecially on 'Toronto' creeping bentgrass greens. In inoculation

tests, cultivars Toronto, Emerald, Highland and Penncross were more

severely affected, but none of the bentgrass cultivars tested were

unaffected. The blighting symptoms on diseased bentgrass plants were

similar to those observed in the field.

Sporulation and conidial morphology in the genera Drechslera,

Bipolaris and the lignicolous Helminthosporium have been shown to be

greatly influenced by culture substrate, culture age, light, temperature

and humidity (Braverman and Graham, 1960; Kafi and Tarr, 1966; Leach,

1967; Ruppel, 1974; Shoemaker, 1962; Tarr and Kafi, 1968). In this

investigation, culture substrate did have major effects on conidial

56

morphology and colony color of the Drechslera catertaria isolate from

bentgrass. The fungus produced an appressed olive-gray colony on

LCH, a whitish to light gray moderately erumpent colony on V-8JA and

a luxuriant slate-gray colony on PDA. Lack of sporulation of

D. catenaria when incubated in the dark has been reported (Shoemaker,

1962; Zeiders, 1976) and regardless of temperature (Leach, 1967). Results

from this study confirm that observation.

Conidia were longer when produced on LCH, but the abruptness of

the taper (AS/BS ratio) did not differ regardless of culture medium.

It is generally true that conidium length is more variable than width

in Drechslera species (Shoemaker, 1962) and this was true with

Drechslera caLenaria. Although the length of conidia varies widely,

the length of the component cells has been reported to be quite a

consistent character for any Drechslera species (Shoemaker, 1962).

Average cell length variation was less for conidia of Q. catenaria

than was conidial length. Basal cell length was also quite consistent.

Therefore, although the length of conidia is generally important,

measurements such as length of basal cell, average cell length and

abruptness of taper appear to be more reliable characters. These

characters should be included in fungal descriptions for separation of

Drechslera species, especially when culture medium is undefined.

Variation among conidial characters was least when conidia were

produced on host tissue as compared with those produced on culture

media. Harding (1975) has strongly recommended the use of a defined

culture medium and controlled environmental conditions for the

57

production of conidia for future comparative work. From this study

of several "Drechslera species, it was concluded that measurements

of conidia are least variable when taken from diseased host tissue

incubated under controlled conditions. These measurements should

preferentially be used in making species determinations based on

conidial dimensions.

The bentgrass isolate DCL caused only necrotic local lesions on

the Festuca and Lo1ium species. Zeiders (1976) found no symptoms on

Festuca or Lolium, but Wilkins (1973) reported a similar flecking

response on resistant genotypes of Lolium perenne when inoculated with

D. catenaria. DCL did cause symptoms on reed canarygrass similar

to those reported by Zeiders (1976) with the distal portions of the

leaves becoming necrotic. However, the D. catenaria isolate from

reed canarygrass (DCZ) demonstrated some host specificity by severely

blighting reed canarygrass but causing little damage to any of the

bentgrasses. Differences in pathogenicity were not observed among DCL

isolates.

Other Drechslera species also varied in host specificity.

D. erythrospila severely attacked the bentgrasses, especially 'Seaside',

as previously reported (Drechsler, 1935; Muse, 1974; Couch, 1973).

Drechslera dictyoides from Festuca arundinacea (DDD) caused severe

damage of that host, but did not induce a net-blotch type symptom

(Drechsler, 1923). The Q. dictyoides isolate from ryegrass (DDP)

infected perennial ryegrass but was less pathogenic on the fescues.

Neither of the D. "dictyoides isolates caused severe damage to any of the

58

~entgrasses. Neither species was pathogenic on Kentucky bluegrass.

This is contrary to previous reports that Q~dictY6ides is the most

important pathogen of bluegrass in Minnesota (Bean and Wilcoxson,

1964; Gibbs and Wilcoxson, 1973; Vargas and Wilcoxson, 1969). This

evidence lends credence to Morrison's (1980) speculation of the mis­

identification of ~. poae as Q. dictyoides on Poa ptatensis.

The fungus isolated from 'Toronto' creeping bentgrass, and shown to

be the pathogen of leaf blight and crown rot, was identified as

Drechslera catenaria (Drechs.) Ito (=Helminthosporium catenarium

Drechs.). The fungus was identified as D. catenaria and not as

Q. dictyoides or any other related Drechslera species for the following

reasons:

a) The conidial characters of the fungus in question are in close

agreement with Drechsler's original description (1923) and with the

conidial morphology and dimensions of the type specimen of H. catenarium

originally submitted by him. The conidia are catenate, with conidia

attached by short secondary conidiophores. Other Drechslera species

including Q. dictyoides and Q. phlei have at times been reported to

produce secondary conidia after long incubation, but the conidia are

commonly sessile (Sharif, 1961; Shoemaker, 1962). The long tapered

conidia of DCL, as well as conidia of the'H~ 'catertarium type specimen,

have hemi-ellipsoidal basal cells with smooth conidial contours;

D. dictyoides has shorter tapered conidia with herni-spherical basal

cells that at times appear inflated with a constriction at the basal

septum (Chidambaram et aI, 1973; Drechsler, 1923; Morrison, 1980).

59

Although D. phlei conidia also taper, they are abruptly enlarged at

the second cell from the base (Shoemaker, 1962). Conidia of

Q. catenaria,D.dictyoides andQ. phlei are described as obclavate

but the degree of taper is consistently greater in D.catenaria.

b) When compared in culture on sucrose proline agar, the bentgrass

isolates and the D.catenaria isolate from reed canarygrass produced a

yellow-orange pigment, while Q. dictyoides isolates produced pink

pigments. These results are identical to Shoemaker's findings (1962).

c) DCL did not extensively colonize Festuca species nor cause

the reticulate symptom pattern that is typical of infection by

Q. dictyoides (Braverman and Graham, 1960; Drechsler, 1923). The

bentgrass isolate only caused minor damage to ryegrass (Lolium perenne).

D. catenaria has not been previously reported as causing a disease

of bentgrass (Agrostis palustris) but has been reported on red top

(Agrostis alba) and associated with the seed of Agrostis vulgaris

(Andersen, 1959; Shoemaker, 1962).

From the morphological and physiological characteristics, it was

concluded that the fungus from 'Toronto' creeping bentgrass was

Drechslera catenaria and that D. catenaria should be retained as a

valid species of Drechslera.

In 1975, Meyer and Turgeon (1975a) reported red leaf spot, caused

by Q. erythrospila, to be a serious disease problem on 'Toronto' (C-15)

creeping bentgrass putting greens in Chicago, Illinois. Symptoms of

red leaf spot are quite similar to those of leaf blight and crown rot,

with the principal symptoms of a drought-stricken appearance of leaves