A STUDY OF VESICULAR-ARBUSCULAR MYCORRHIZAL(VAM) FUNGI ONCOOL-SEASON TURFGRASS

A Project Report

Presented to the Faculty of the Graduate School

of Cornell University

in Partial Fulfillment of the Requirements for the Degree of

Master of Professional Studies

by

James B. Frank

May 1984

Members of Special Committee: A. Martin PetrovicK. W. MudgeR. Smil ey

A STUDY OF VESICULAR-ARBUSCULAR MYCORRHIZAL(VAM) FUNGI ONCOOL-SEASON TURFGRASS

A Project ReportPresented to the Faculty of the Graduate School

of Cornell Universityin Partial Fulfillment of the Requirements for the Degree of

Master of Professional Studies

by

James B. FrankMay 1984

Members of Special Committee: A. Martin PetrovicK. W. MudgeR. Smil ey

ABSTRACTGrowth effects of YA mycorrhiza on a variety of plants

has been well documented. However, this area of researchhas received little attention in relationship to highlymaintained cool-season turfgrasses. Before studying YAmycorrhiza (YAM) growth effects on turfgrass, it wasnecessary to examine the presence or absence (or extent)of this association under field conditions.

A field survey was conducted to determine the level ofYAM infection of four turfgrass genera and severalcultivars, including: Kentucky bluegrass (Poa pratensisL. cvs. Park, Ram I, Bonnieblue, Touchdown); chewingsfescue (Festuca rubra ~. commutata Gaud. cv.Wintergreen); hard fescue (Festuca ovina ~. duriusculaL. Koch cv. Scaldis); perennial ryegrass (Lolium perenneL. cv. Citation) ; and creeping bentgrass (Agrostispalustris Huds. cv. Seaside). It was concluded that YAMfungi are indigenous in intensively managed cool-seasonturfgrasses. Infections by these fungal symbiontsoccurred on at least 50 % of the root systems examined.YAM infection ranged from a low of 58% on two Kentuckybluegrass cultivars to a high of 77% for Citation

perennial ryegrass.Two greenhouse studies were conducted to examine the

effects of YAM fungi on the growth of Citation perennial

ryegrass.

It was found that the growth of perennial ryegrass for

77 days in a phosphorus-deficient Windsor fine sandy loam

(mixed mesic, Uditsamment) was increased by VAM

inoculation and by supplemental phosphorus (by 287. and

907., respectively). However, with supplemental

phosphorus, VAM inoculation depressed the growth by 327..

Supplemental phosphorus also reduced VA mycorrhizal

infection by 377.. Although a mixture of four VA

mycorrhizal species (Glomus macrocarpus var. macrocarpus,

~. mosseae, Q. fasicu1atus, Gigaspora margarita) caused a

507. higher infection of Ci ta tion perennial ryegras s root

system, compared to the Glomus macrocarpus var.

macrocarpus alone, there were no corresponding growth

differences. Nutrient analysis of shoots showed that

applied phosphorus caused a 587. increase in phosphorus

uptake on a 7. dry weight basis. However, VAM-inocu1ated

plants did not have an increased concentration of

phosphorus in the clippings, as compared to the non-YAM

inoculated plants.

The effects of VAM fungal infections as a function of

different inoculum placement levels within a soil profile

on Citation perenia1 ryegrass was also studied under

conditions of low soil phosphorus. Inoculum was placed at

various depths within the soil columns prior to seeding

thereby delaying the time at which the plants became

infected. It was found that the sooner the host roots

became infected with the VAM fungus the more rapid was the

resulting growth enhancement. VAM infection occurred even

at the deepest inoculum placement (20.3cm) and was always

confined to a narrow zone surrounding the inoculum layer.

Further research is needed on the interaction of VA

mycorrhiza with shoot and root growth at several levels of

supplemental phosphorus.

BIOGRAPHICAL SKETCH

James B. Frank was born in Albany, New York on August31, 1952. He graduated from Albany High School in thespring of 1970. The author received a Bachelor of Arts inPsychology from the State University of New York Collegeat Brockport in 1974. He was employed on the grounds I

crew at Irondequoit Country Club in Rochester, New Yorkand in 1977 he became their assistant golf coursesuperintendent. Aspiring to become a golf coursesuperintendent, the author entered graduate school atCornell University, where he became enrolled in theDepartment of Floriculture and Ornamental Horticulture in

September, 1981.

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DEDICATIONTo my wife Robin who lovingly supported her husband during

the trials and

tribulations of being a graduate student.

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ACKNOWLEDGEMENTS

The author wishes to thank Dr. A.M. Petrovic, chairman

of his graduate committee, for providing guidance and

input into the art of scientific experimenta tion. The

author also wishes to thank his minor committee members,

Dr. K. Mudge and Dr. R. Smiley, for their assistance in

his research project. Special thanks are also given to

Dr. W. Sinclair, who although had no formal commitment to

the authors' graduate committee, offered much of his time

and insight into the study of VA Mycorrhizae.

The author also wishes to thank Mr. Howard Pidduck for

his help and assistance in the physical set up of the

authors' greenhouse experiment. Appreciation goes out to

Mr. Ronald White for his willingness to go above and

beyond the call of duty to provide assistance in many

areas, but especially in utilizing his knowledge of

computer skills. Appreciation also goes out to Ms.

Melissa Craven Fowler for assisting the author in the area

of microphotography. The author also wishes to thank Mr.

Robert Whipple who provided assistance in analyzing the

nutrient content of foliar tissue samples.

It would be negligent for the author not to mention all

the help received from the Department's Greenhouse Staff,

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who provided their sincere cooperation and moral support

given by fellow graduate students.

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TABLE OF CONTENTS

BIOGRAPHICAL SKETCHDEDICATIONACKNOWLEDGEMENTS

. ii

iii.. i v

Chapter

1. LITERATURE REVIEW .In troduc tion ..Biology of VAM Fungi .

VAM MorphologyVAM Spore Dispersal .....•..

Effects of VAM on Plant Pathogens andPlant Growth .•..........

VAM Interactions With Plant Pathogens .Enhanced Nutrient Uptake .Growth Enhancement .VAM Induced Plant Growth Depressions

Mycorrhizal Associations With Turfgrasses

1

1224

557

. 10

. 13•• 17

II. FIELD SURVEY OF VAM ASSOCIATIONS ONMAINTAINED TURFGRASSES . 19

. 19•. 20

. 20. . . . . 20

• • 23•• 24

...... 25• • 26

Introduction .Methods and Materials

Turfgrasses Sampled .Site Characteristics .Root Sampling •.Quantification of VAM Infection

Results .....Discussion .....•.....

III. GREENHOUSE STUDY . 30

• 30• • 31•. 31

. . . . . 35...... 39

39..... 39

• • 40. 42

Introduction ..•...Methods and Materials

Experimental Design ..VAM InoculumSelection of a TurfgrassSoil CharacteristicsSoil Column Preparation .Greenhouse Bench Construction •.Seeding and Fertilization

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Other Maintenance Considerations 43Parameters Evaluated 44Data Analysis ...•...•.... 45

Results for Experiment I 46% Germination 46Growth Effects ........•.. 46VA Mycorrhizal Infection Assessment .. 49Nutrient Analysis of Clippings . 50

Re suI ts for Experiment II 537. Germination 53Growth Effects 54VA Mycorrhizal Infection Assessment .. 64Nutrient Analysis of Clippings .. 69

Discussion •..•...•...••.. 71IV. CONCLUSIONS •... .. 78

AppendixMICROPHOTOGRAPHY OF VA MYCORRHIZAL STRUCTURES FOUND

IN CITATION PERENNIAL RYEGRASS ROOTsY STEM S •••••••••••••••••• 81

BIBLIOGRAPHY ...................

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•• 86

LIST OF TABLES

Table

1. Chemical analysis of field survey soil •... 21

2. 1982 fungicide program for Seaside creepingbentgrass 22

3. Mean % VAM infection levels of Field Surveyedturfgrasses during May and October 1982 .... 26

4.

5.

6.

Mean % VAM infection levels of Kentuckybluegrass treated with fungicides

Summary of treatments for Experiment I .

Summary of treatments for Experiment II

• 27

• 32

• 34

7. Source of VAM original isolates . 35

8. Chemical analysis of soil used in ExperimentI, II 36

9. Effects of phosphorus and VA mycorrhiza typeon germination of Citation perennialryegrass for Experiment I 46

10. Sumary of analysis of variance of cumulativeclipping yields for Experiment I 49

11. Effects of soil phosphorus and VA mycorrhizaltype on colonization of Citation perennialryegrass root system by VA mycorrhiza ..... 50

12. Effects of phosphorus and VA mycorrhiza typeon clipping nutrient content of Citationperennial ryegrass ...•.....•.... 51

13. Effects of inoculum placement and VAmycorrhiza type on total nutrient uptake 77days following germination of Citationperennial ryegrass for Experiment I ....•. 53

14. Effects of inoculum placement and VAmycorrhiza type on germination of Citationperennial ryegrass for Experiment II .. 54

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15. Summary of analysis of variance of cumulativeclipping yields for Experiment II 63

16. Effects of inoculum placement and VAmycorrhizal type on the averagecolonization of Citation perennial ryegrassroots by VA mycorrhiza for Experiment II . 64

17. Effects of inoculum placement and VAmycorrhiza type on clipping and verdurenutrient content 77 days followinggermination of Citation perennial ryegrassfor experiment II .••.•.....•.... 69

18. Effects of inoculum placement and VAmycorrhiza type on total nutrient uptake 77days following germination of Citationperennial ryegrass for Experiment II .. 70

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LIST OF FIGURES

Figure

1. Effects of phosphorus and single and multiplespecies of VA mycorrhiza on shoot growthand verdure of Citation perennial ryegrassfor Experiment I ........•. 48

2. Effects of mixed VA mycorrhizal species atfour different placement levels oncumulative shoot growth and verdure yieldof Citation perennial ryegrass forExperiment II 55

3. Effects of pure VA mycorrhizal species at fourdifferent placement levels on cumulativeshoot growth and verdure yields of Citationperennial ryegrass for Experiment II .. 56

4. Cumulative clipping yields for theuninoculated check treatments at differentplacement levels for Experiment II 57

5. Effects of surface placed single and multiplespecies of VA mycorrhiza on cumulativeshoot growth and verdure yields of Citationperennial ryegrass for Experiment II .. 59

6. Effects of 5.1cm placed single and multiple VAmycorrhiza species on cumulative shootgrowth and verdure yields of Citationperennial ryegrass for Experiment II ..... 60

7. Effects of 10.2cm placed single and multiplespecies of VA mycorrhiza inoculum oncumulative shoot growth and verdure yieldsof Citation perennial ryegrass forExperiment II 61

8. Effects of 20.3cm placed single and multiplespecies of VA mycorrhiza inoculum oncumulative shoot growth and verdure yieldsof Citation perennial. ryegrass forExperiment II 62

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9. Effects of inoculum placement on VA mycorrhizacolonization of Citation perennial ryegrassroot system within the soil profile ... 65

10. Effects of surface placed VA mycorrhizainoculum on VA mycorrhiza colonization ofCitation perennial ryegrass root systemwithin the soil profile ..... 67

11. Effects of s.lcm placed VA mycorrhiza inoculumon VA mycorrhiza colonization of Citationperennial ryegrass root system within thesoil profile 67

12. Effects of 10.2cm placed VA mycorrhizainoculum on VA mycorrhiza colonization ofCitation perennial ryegrass root systemwithin the soil profile 68

13. Effects of 20.3cm placed VA mycorrhizainoculum on VA mycorrhiza colonization ofCitation perennial ryegrass root systemwithin the soil profile 68

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Chapter ILITERATURE REVIEW

1.1 INTRODUCTIONThe term mycorrhiza has its literal translation coming

from the two Greek words "myco", meaning fungus, and"rhiza" , meaning root. Endomycorrhiza1 fungi are groupedwithin the class Zygomycotinia and the family Endogonaceae(Mosse, 1981). Mycorrhizae fungi are obligate1y parasiticsoil fungi that colonize and inhabit roots in a symbioticrelationship with the host plant (Mosse, 1981). Many ofthe endomycorrhiza1 fungi are also referred to asvesicu1ar-arbuscu1ar mycorrhizae (VAM) because of thevesicles and arbuscu1es that they produce (Maronek et al.,

1980) .In natural ecosystems VAM fungal associations commonly

occur in numerous plant families (Mosse, 1981). The VAMfungal dependency by plants can be viewed not only inabsolute values but also in terms of a continuum. It hasbeen reported that plant species which either lack or havepoorly developed root hairs are more dependent onmycorrhizae fungi for accumulation of phosphorus and othernutrients than are those plant families that have a welldeveloped root hair system (Baylis, 1970, 1972).

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Since the world wide phosphorus fertilizer reserves arediminishing it may become necessary, in the near future,to take advantage of low levels of otherwise unavailablesoil phosphorus with the aid of YAM fungi (Maronek et al.,1981) .

1.2 BIOLOGY OF YAM FUNGI1.2.1 YAM MorphologyDormant YAM propagules within the soil germinate inresponse to factors emanating from passing roots. Thegermination results in production of an appressorium, fromwhich an infection peg can penetrate the epidermal tissueof plant roots (Mosse, 1973), Production of hyphaefollows, and they grow intercellulary into the root cortexwithout colonizing the root tip (Mosse, 1973). YAM fungido not invade the secondary cortical tissue and the mainstructural roots of perennial plants (Mosse, 1981).

An extensive network of external mycelium also extendsinto the surrounding soil (Gerdeman, 1975). The externalmycelium is non-septate and is approximately 8 to 12um indiameter, with main branches reaching up to 20um indiameter (Mosse, 1981). Hyphae of YAM fungi are usuallynon-septate, but when growing conditions of the host plantare unfavorable or the hyphae are dying, septa may beformed (Gerdeman, 1975). Because these infections and

subsequent colonizations produce few chang'es in external

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root morphology, the YAM fungus within the root can onlybe seen microscopically, after the root is cleared andstained (Mosse, 1973).

Arbuscules of the YAM fungi are formed within the cellsof the root cortex. The arbuscules are the analogues tohaustoria. As they are formed, starch within the invadedcells disappears, cell nuclei enlarge, and then oftendivide (Gerdeman, 1975). Arbuscules are the result ofrepeated dichotomouspenetrated the host

branching ofcells (Mosse,

hyphae1973) .

that havePictorially,

they resemble the silhouette of a defoliated tree

(Gerdeman, 1975). Arbuscules usually die within one tothree weeks after they are formed. As the fungal cell

walls collapse, it is thought that some of the arbuscularcontents are translocated to various parts of the hyphalnetwork, while other contents remain within the host

cytoplasm (Mosse, 1981).Terminal saclike structures, called vesicles, are often

formed at hyphal tips and contain

serve as storage organs. Aslipid droplets whichthe root undergoes

decortication of its primary cortex, the vesicles go fromthe root to the soil where they may germinate and serve asvegatative propagules of the fungus. The occurrence ofvesicles appear s to be somewhat seasonal. They usuallybecome more numerous late in the growing season as thehost plant reaches maturity (Mosse, 1981).

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Some YAM fungal species form spores and sporocarps,whereas other species do not. Spore survival within thesoil has been known to last for several months and evenyears. Spore diameters may range from less than 100um to600um. These fungal spores are some of the largest thatare known in the fungal kingdom. Because of their size,spores can be extracted from soil samples through variouswet sieving procedures, which are similar to those used toextract nematode eggs. Classification of YAM fungi togenus and species is based on spore characteristics(Mosse, 1981>.

In temperate climates there is a seasonal fluctuationin YAM fungi sporulation. In July, a large increase inspore production begins, with a slow decrease fromSeptember until December, at which time spore productionceases (Hayman, 1970).

1.2.2 YAM Spore DispersalYAM fungi are worldwide in distribution, being found

from such diverse climates as the tropics to the arctic.Yery few natural plant communities do not contain YAMassociations (Gerdeman, 1975). Dispersal of the sporesdoes not occur through air, but rather in drainage wateras well as any means of soil movement such as windblownsoil particles and erosion. Dispersal is even thought tooccur via animals since YAM fungal spores have been. found

in their stomachs (Mosse, 1981).

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1.3 EFFECTS OF VAM ON PLANT PATHOGENS AND PLANT GROWTHVAM fungi have been shown to have effects on plant

growth. Interactions between VAM fungi and pathogenicfungi also occur.

1.3.1 VAM Interactions With Plant PathogensInteractions between VAM fungi and plant pathogens have

received much attention. Since mycorrhizal infections are1 imited to plant roots, most of the research concerningVAM enhancement of disease resistance has concentrated ondiseases caused by soil-borne pathogens. In general, VAMinfected plants have less disease damage, lower diseaseincidence, and show inhibited pathogenic colonization fromsoil-borne pathogens, as compared to non-endomycorrhiza1plants (Dehne, 1982). Increased plant vigor may result ina higher level of resistance to some soil-borne pathogens(Dehne, 1982). Endomycorrhiza1 tomato plants were shownto have lower amounts of mycelial growth from Fusariumoxysporum f. sp. 1ycopersici than non-endomycorrhiza1plants (Dehne and Schoenbeck, 1979). In addition, damagecaused by soil-borne nematodes has been observed to besignificantly less severe on VAM colonized plants (Dehne,

1982) .Although, this generalization is true in many

situations, there are exceptions in which the VAM fungus.has been reported to decrease disease resistance. The

are also capable of influencing localizedalterations resulting in both changes in

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disease increase is thought to be a result of YAM infectedplants having elevated nutritional levels in nutrientdeficient soils, which results in both increased plantgrowth and elevated pathogenic activity (Dehne, 1982). Ithas been shown, for instance, that cotton plantsinoculated with YAM fungi had higher growth levels thannon-YAM cotton plants. The severity of Yerticillium wilt,a soil-borne disease, was also greater on the mycorrhizalplants. Subsequent additions of phosphorus to both YAMplants and non-YAM plants also caused an increase in thisvascular disease (Davis et al., 1979).

Additionally, diseases of shoots and leaves aresometimes enhanced by the presence of YAM. It has beenproposed that the elevated nutrition of YAM plants couldbe directly responsible for the higher suceptibility ofshoots and leaves to fungal and viral pathogens (Dehne,

1982) .Although YAM fungi are in some instances capable of

retarding pathogenic colonization in roots, thiscapability is restricted to the actual sites of YAMinfections. The diseases which are enhanced by thepresence of the mycorrhizal fungus, are systemicallytransmitted through the plants vascular system (Dehne,

1982) .YAM fungi

physiological

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root morphology and disease resistance (Dehne, 1982). Ithas been shown that YAM infected Tomato roots were morelignified than non-YAM roots, especially in stelar tissue(Dehne, 1982). This may be why YAM infections are limitedto cortical tissue and could explain the enhancedresistance of YAM colonized plants to soil-bornepathogens, since lignin acts as a barrier to fungalpenetration (Dehne, 1982). It is also interesting to notethat YAM colonized roots have been shown to have higherchitinolytic enzyme activity, which is thought to beeffective as a defense mechanism against pathogenic attack(Dehne, 1982).

been attributed1.3.2 Enhanced Nutrient Uptake

Plant growth enhancement by YAM hasmainly to increased phosphorus uptake, since

plantsinfectednon-YAM

endomycorrhizal plants typically have greaterconcentrations of phosphorus than non-endomycorrhizalplants (Mosse, 1981). The elevated phosphorus levels havebeen attributed to an increased efficiency of absorptionof phosphorus by hyphae of YAM fungi (Sanders and Tinker,1971). However, there is also evidence that YAM fungienhance the uptake of other nutrients such as Zn, S, Sr,

and eu (Gerdeman, 1975).Phosphorous uptake by

ordinarily occurs within a 1-2mm zone surrounding the

is highlythe most

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rootlet (Rhodes and Gerdeman, 1975). Regardless ofwhether plants are infected with VAM fungi, generally onlythe soluble fraction from soil is absorbed (Gerdeman,1975) .

Soil phosphorus forms consist of two major groups:organic and inorganic. The organic forms are furthercategorized into those containing calcium and thosecontaining iron and aluminum. Those sources that containcalcium in increasing order of solubility, include, florapetite, carbonate apetite, oxy apetite, tricalciumphosphate, dicalcium phosphate, and monocalcium phosphate.Those sources containing iron and aluminum are usuallyless soluble than the calcium forms. Although the organicgroup has received much less attention than the inorganicgroup it accounts for a considerable amount of thephosphorus within the soil. The organic forms can becategorized as follows: phytin and phytin derivatives;nucleic acids; and phospholipids.

The availablity of phosphorus in soildependent on the soil pH. The H2P04- ion is

available form under slightly acid conditions and the

HP04= ion is most available under slightly alkaline

conditions (Brady, 1974).

Phosphorus uptake is limited by the slow conversion of

insoluble phosphorus into the soluble form. In non-YAMinfected plants the 1-2mm soil zone around roots readily

linear extension of

becomes depletedexplored by thelimited by slow

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of phosphorus.root hairs.

ThisUptake

zone of soil isis then further

roots (Rhodes andGerdeman, 1975). In YAM infected plants, however,enhanced phosphorus uptake is attributed to the extensionof mycelium well beyond the zone of root hair penetration.The mycorrhizal mycelium can extend at least 7cm from theroot surface (Rhodes and Gerdeman, 1975). In otherwords,the external mycelium serves to increase the effectiveroot surface area for nutrient uptake (Bowen et al., 1975,Mosse, 1981, Sanders and Tinker, 1981).

Once phosphorus uptake by the YAM mycelium occuurs itmust be translocated to the root. Cox et a1. (1975)described the common occurrance of phosphate granules,O.lum to O.2um in diameter, within YAM fungal cellvacuoles. They believe that these granules are in anequilibrium with the vacuolar sap, which is saturated withpolyphosphates. The vacuoles, mobile via cytoplasmicstreaming, function to translocate phosphorus within thefungal body. It has been observed that the finearbuscular branches within host root cortical cellscontain vacuoles without phosphorus granules. Thissuggests that the fine arbuscular branches are thelocation at which polyphosphates are converted fortransfer into the host plant (Cox et al., 1975).

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1.3.3 Growth Enhancement

There are several proposed mechanisms by which YAM

fungi are able to enhance the growth of plants. This

includes the theory that YAM fungi decrease the resistance

to water transport (Safir et a1., 1971). Gerdeman (1975)

believes the mechanism is associated with enhanced uptake

of several minor nutrients such as Zn, S, Sr, and Cu.

However, increased uptake of phosphorus, especially in

low phosphorus soils, by YAM infected plants, is

considered to be the major factor contributing to

increased growth over non-YAM infected plants. It has

been found that YAM fungi and added phosphorus have

similar effects on plant growth (Mosse, 1981). There are

smaller growth differences between YAM infected plants and

non-infected plants as soil phosphorus levels become more

sufficient. When soil phosphorus reaches optimum and

above optimum levels, YAM-induced growth depressions have

been found (Mosse, 1981).Clearly the presence of sufficient levels of phosphorus

is important to plant growth. YAM fungi can make up for a

phosphorus deficit for a wide range of plant families.

Within the family Magno1iacae, YAM infected Magnolia trees

had nearly a two-fold increase in height over non-YAM

Magnolia in low phosphorus soil (Maronek et al., 1980).

In the Roseacae family, a significant positive growth

effect was also achieved with YAM-infected apple tree~

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over non-infected ones when grown under conditions of lowsoil phosphorus. This occurred even when phosphorus wasadded to otherwise low phosphorus soil (Plenchette et al.,1981). Also within this family YAM inoculations enhancedthe growth of peach trees as much as did optimum levels ofsoil phosphorus (Strobel et a1., 1982). Similar resultswere found in the Gramineae family with ryegrass,cocksfoot, and sweet vernal (Crush, 1973).

Many plant families as well as cultivars have largedifferences in their capability to extract phosphorus fromthe same soil. Certain species will show symptoms ofphosphorus deficiencies, while others will not. This isalso true for the response to YAM infections among plantspecies (Mosse, 1973>' For example, in an agriculturalsoil Liquidambar, onions, and Coprosma had as much as tenfold increases in growth with YAM infection over non-YAMcontrols. In contrast, Nordus stricta and Fuchsia hadlittle change in growth even when additional phosphoruswas added. However, in less fertile sand, Nordus growthwas much improved by YAM associations (Mosse, 1973). Ithas been found that species variability of YAM dependencehas been related to root hair development (Baylis, 1970,1972). Therefore, the major factors determining plantresponse to YAM fungi are the plant species and the levelof available soil phosphorus.

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The critical level of soil phosphorus for each of

several plant species was determined by Cooper (1975).

When phosphorus was above this critical level, growth

occurred equally between plants that were with and without

the YAM fungi. Below the critical phosphorus level there

was an increasing dependency on the symbiosis. Plants

grown near the critical phosphorus level, in which there

wa s only a slight YAM dependency, were made more YAM

dependent by adding a complete nutrient solution minus

phosphorus. The greater YAM dependency was attributed to

the fact that there was a greater need for more phosphorus

created by the nutrient imbalance.

The transfer of nutrients from the soil to the fungus

and then on to the host plant is a complex system. It has

been determined that YAM infection levels were higher when

organic sources of phosphorus (phyate) were available

rather than when inorganic sources were most available

(Allen et al., 1981). However, conversions of phosphorus

from inorganic forms to organic forms within leaves

consistantly increased with YAM infected plants,

suggesting enhanced levels of photophosphorylation

(Simonis and Urback, 1973>' Allen et a1. (1981) showed

that photosynthesis increased in Bouteloua gracilis as the

level of mycorrhizal infections increased.

Enhanced uptake of minor nutrients (Zn, S, Sr, and Cu)

is also given as a possible mechanism for YAM induced

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plant growth enhancement. However, the research on this

concept has yielded inconsistant results (Gerdeman, 1975;

Mosse, 1973, 1981; Maronek et aI., 1980). Some

experiments show higher concentrations of these elements

in VAM colonized plants while other research shows the

opposite. For instance Benson and Covey (1976) found that

VA mycorrhiza increased shoot weight of apple trees but

had no effect on zinc concentrations. On the other hand

Gilmore (1971) found that VA mycorrhizal inoculations were

able to correct a zinc deficiency in peach trees.

Another proposed mechanism for YAM-induced growth

enhancement is decreased resistance of water transport.

Sa fir et a1. (1971) found that the decreased resistance to

water transport in VAM infected plants was the means by

which VAM fungi are able to increase plant growth over

non-YAM infected plants. However, it was later shown by

Sa fir et a1. (1972) tha t an inc rease in the nutrient

status of non-YAM plants alone could account for the

decreased resistance to water transport. They conclude

that the elevated nutrient status of the VAM plants caused

the decreased resistance to water transport.

1.3.4 VAM Induced Plant Growth Depressions

Even though it is common to find VAM enhanced plant

growth, growth depressions are also very common. The

plant growth depression seems to occur only when soil

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phosphorus is either non-limiting to plant growth or inexcessive amounts (Mosse, 1973, 1981; Crush, 1973; Strobelet a1., 1982). Mosse (1973) believes the plant growthdepression phenomenon is due to a build up of a toxiclevel of phosphorus. Other research (Cox et a1., 1975)has contradicted this theory, by showing that the growthdepressions are related to the increased translocation ofcarbon from host plant tissue to the YAM fungus. Resultsof a more recent experiment (Buwa1da et a1., 1982)supports the increased carbon translocation theory. Thesefindings indicate that phosphorus toxicity is not aprecursor to YAM associated growth depressions with highsoil phosphorus levels. Buwa1da et a1. proposed that thecompetition between the host plant and the YAM fungus forphotosynthetically derived carbon is the mechanism forgrowth depressions.

Development of this theory is based on the followingresults for YAM plants: they have reduced levels ofcarbon; a significantly lower amount of total oxidizablesugar; a lower soluble sugar content; and a lower carbonto nitrogen ratio. The lower levels of soluble sugars areespecially important because these sources of carbonrepresent the non-structural carbohydrates which are theprimary stored material used for plant growth (Buwa1da andGoh, 1981).

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Others have confirmed this theory that the mechanism ofgrowth depression is a result of the YAM fungus acting asa carbon sink under high levels of soil phosphorus. Hoand Trappe (1973) found 14C in the spores of YAM fungifrom plants receiving 14C02. Labled photosynthetic carbonwa s observed to be translocated from the host to thefungal mycelium (Cox et al., 1975).

The amount of phosphorus within the host root maytherefore influence ATP levels within the YAM fungus.This in turn effects phosphorylation by enhancing fungalcompetition for active transport of carbon to the fungusfrom the plant (Buwalda and Goh, 1982).

Mosse (1973) interestingly noted that whenever growthdepressions occurred, there was also a decrease in YAMarbuscules and in general a substantially lower level ofintracellular infections.

Thus it appears that mycorrhizal associations areself-regulating. When there is no longer any advantage inthe association for plants, they develop an immunity toYAM fungi. Also, the phosphorus levels at which YAMinfections begin to decrease is the same point at whichgrowth depressions are noted (Mosse, 1981).

The level of infection related to an ecological niche

can be explained in physiological terms.Sudangrass (Ratnayke et a1., 1978), it

In a study withwas found that

amounts of soluble amino acids and.reducing sugars in root

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exudates were higher under low soil phosphorus than underhigh phosphorus levels. The amounts of these substancesin the root exudates was independent of the concentrationof the same substances within the roots themselves. Theyalso observed a high correlation between degree ofexudation and root membrane permeability, which was alsorelated to a decrease in the phospholipid content withinthe root tissue. Simon (1974) also found, underconditions of low soil phosphorus, a sharp decline in thephospholipid layer within root membranes, leading toincreased membrane permeability.

When available soil pho'sphorus increases, both rootmembrane permeability and root exudation decrease. Thisis suggested as a possible mechanism for phosphoruscontrol of YAM infection. Under conditions of highavailable soil phosphorus, the metabolites from the rootsare not exuded in sufficient amounts to initiate YAMinfections. Under low levels of available soilphosphorus, however, metabolites are exuded in necessaryamounts to support initial growth and development ofgerminating YAM spores, thereby overcoming the soilfungistasis. Subsequent infection and colonization by YAMfungi, leads to improved phosphorus levels within theplant roots resulting in decreased metabolic exudations(Ratnayke et al., 1978).

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After a root has been colonized, the YAM fungus can

fulfill its needs for carbon by increasing the surface

area within the host cells by forming the fungal

arbuscules (Ratnayke et a1., 1978). Therefore,

establishment of the YAM fungus within a root requires a

minimal level of carbohydrates in root exudates. Thus,

beyond the initial infection, the level of colonization is

controled by other aspects. For instance, it was found

that YAM infections led to a decrease in both reducing

sugars and total sugars within the root (Azcon and Ocampo,

1981). A high correlation was observed between a decrease

in sugars within the root and an increased level of

mycorrhizal infections.

1.4 MYCORRHIZAL ASSOCIATIONS WITH TURFGRASSES

Although there have been several studies involving YAM

associations in the Graminaea family, there has been

little work done with YAM on maintained turfgrasses.

For instance, Powell (1975) found that in the hill and

high country soils in New Zealand mycorrhizal infection

wa s quite common on a pasture type Lolium perenne L.

Powell (1977) later found that pasture type perennial

ryegrass exhibited increased growth with YAM at low soil

phosphorus levels.

More recently, Lambert and Cole (1980), while working

on a low fertile acidic mine spoil soil, found the growth

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of Pennfine perennial ryegrass increased by 607. wheninfected with mycorrhizal fungi. However, this increasewas not found to be statistically significant, which wasmainly attributed to replication variation and otherlimiting environmental effects not influenced by themycorrhizal fungus.

A highly maintained turfgrass is typified by a golfcourse putting green. Rhodes and Larsen (1981) foundlimited evidence that certain fungicides, when appliedearly in the growing season, reduced mycorrhizaldevelopment on creeping bentgrass (Agrostis palustrisHud s.).

Chapter IIFIELD SURVEY OF VAM ASSOCIATIONS ON MAINTAINED TURFGRASSES

2.1 INTRODUCTIONNumerous plant families have been cited as having VAM

relationships (Mosse, 1981). Although VAM occurrance ingrasses has long been known to exist, most field surveyson noncerea1 grasses have been concerned mainly withmycorrhizal associations in either pasture lands ornatural ecosystems. There is little evidence which firmlyestablishes that VAM fungal associations occur onintensively managed turfgrass. Rhodes and Larsen (1981)observed that creeping bentgrass golf greens had about 507-

of the root length colonized by several Glomus sp. of YAM.Fungicides are used on turfgrasses as part of a disease

control program, but there can also be deleterious sideeffects from fungicide use (Smiley, 1981).

The fungicides ch10rotha10ni1, benomy1, and iprodionehave been shown to have either detrimental effects on YAMcolonization (Rhodes and Larsen, 1981) or, in someinstances, actually enhance YAM colonization (Nemec,1979). The enhancement was thought to result fromfungicidal inhibition of YAM fungal antagonists.

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The objectives of this field survey are two-fold.

First, to firmly establish if there exists an association

between cool season turfgrasses and YAM fungi. Secondly,

to determine if there is an increase or decrease in YAM

colonization of turfgrass roots during the course of a

growing season, in which fungicides are used.

2.2 METHODS AND MATERIALS

2.2.1 Turfgrasses Sampled

This field survey consisted of studying the YAM

infection level of the following four turfgrass genera and

cuI tivar s:. Kentucky bluegrass (Poa pratensis L. cvs.

Park, Ram I, Bonnieblue, Touchdown); chewings fescue

(Festuca rubra ssp. commutata Gaud. cv. Wintergreen);

hard fescue (Festuca ovina ssp. duriuscula (L) Koch cv.

Scaldis); perennial ryegrass (Lolium perenne L. cv.

Citation); and creeping bentgrass (Agrostis palustris

Huds. cv. Seaside).

Roots from an ongoing longterm fungicide study on a

stand of Kentucky bluegrasses were also sampled to

determine fungicide effects on YAM colonization over the

course of a growing season.

2.2.2 Site CharacteristicsAll cuI tivar s sampled were established on an Arkport

sandy loam soil at th~ Cornell University Turfgrass Field

Research Laboratory.

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The soil consisted of about 617.sand, 307.silt, and 87.clay. Results of the soil chemicalanalysis, conducted by the Agronomy Department SoilTesting Laboratory, Cornell University, are shown in Table1.

TABLE 1Chemical analysis of field survey soil

pH4.8

Ex. H me/100g.10

P K Mg Ca---------------PPM-----------

2.4 19 13 170

The perennial ryegrass area was established in 1979,maintained at a cutting height of 3.8cm, and has beenfertilized with urea at a rate of 190 kg N/ha/yr. TheKentucky bluegrasses and fescues were established in 1976and were maintained at a cutting height of 3.8cm. From

1976 to 1979 they were fertilized at a rate of 47.5 kgN/ha/yr, applied as 13-13-13 fertilizer. Since 1979 theywere fertilized with urea at a rate of 47.5 kg N/ha/yr.

The Seaside creeping bentgrass plot was established in1978, maintained at a cutting height of about 1cm, andfertilized with urea at a rate of 190 kg N/ha/yr.

The ongoing fungicide study was established in 1979from sod of a blend of 'Victa', 'Vantage', 'Windsor' and'Merit' Kentucky bluegrass (Poa pratensis L.). These

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plots were maintained at a cutting height of 3.8cm, and

fertilized with urea at a rate of 190 kg N/ha/yr. Root

samples were taken from the following fungicide treated

areas: triadimefon applied at 2.45 kg active

ingredient/ha/yr; benomy1 applied at 4.92 kg active

ingredient/ha/yr; and a non-treated control.

Table 2 shows the fungicide program for 1982. The same

basic program was utilized in 1980 and 1981, with only

minor alterations. No other pesticides were used in 1980

and 1981. In 1982 the insecticide isofenphos, at 0.9 kg

active ingredient/ha/yr, was used on the Seaside creeping

bentgrass to control European Chafer. No herbicides were

used at any time.

TABLE 2

1982 fungicide program for Seaside creeping bentgrass

Target DiseaseActive

Ingredient(AI)

carbendazimmanebch10rotha1oni1*methy1thiophanate*mancozebbenomy1captancadminate

Leaf SpotDollar Spot-Brown PatchDollar SpotDollar Spot-Brown PatchDollar Spot-Brown PatchDollar Spot-Brown PatchDollar Spot-Brown PatchSnow Mold

AI **kg/ha/yr

2.632.911.113.754.91.50.33.1

**=app1ied in more than one application*=these two were in a combination product

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The only pesticide used on the perennial ryegrass overthe three year period of 1980 to 1982 was benomyl. It wasapplied at 3 kg/ha/yr for dollar spot and brown patchcontrol. The fine fescues received no pesticideapplications. In 1981 and 1982 no pesticides were used onthe Kentucky bluegrasses. However, the followingpesticides were applied in 1980: 2,4-0 for broadleaf weedcontrol (1.1 kg/ha/yr); maneb for controling dollar spotand brown patch <19.8 kg/ha/yr); and benomyl forcontrolling dollar spot and brown patch (1.5 kg/ha/yr).

2.2.3 Root SamplingRoots of the cultivars named above and the long term

fungicide study were sampled on 28 May 1982 and again on14 October 1982. The 28 May sampling was made prior tothe first fungicide application whereas, the 14 Octobersampling was taken after the last fungicide applicationfrom the longterm fungicide study. Three samples weretaken from one plot of each turfgrass on each date. A1.9cm diameter soil sampling tool was used to extract asample approximately 15cm deep. The thatch layer andverdure was removed, the soil were washed from the roots,and then immediately fixed for later quantification of YAMfungal infection levels. The fixing solution, commonlyreferred to as FAA, consisted of formalin, glacial aceticacid, and 507. ethanol in a 13:5:200 ratio, respectively

(Philips and Hayman, 1970).

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2.2.4 Quantification of YAM Infection

Prior to staining and quantification, roots were

subsampled by random selection from the FAA fixed samples

and were cut into 1 to 2cm pieces. The procedure of

Philips and Hayman (1970) was used to prepare the roots

for quantification. Briefly this procedure involved

removing the host cytoplasm by simmering the roots in 107.

KOH, then acidifying them in a dilute 17. solution of HCl,

which was then followed by staining the roots with 0.057.

trypan blue solution.

To determine the proportions of infected tissue, the

gridline-intercept method of Giovannetti and Mosse (1980)

was used. A 60X compound microscope was used for

determination of root intercepts and the presence of YAM

structures. Determination of infection at root intercepts

was based on the presence of either vesicles, arbuscules,

or YAM hyphae (see Appendix) . Quantification was,

therefore, based on the ratio of the number of YAM

infection points per number of root intercepts, which is

given as 7. infection. No attempt was made to identify the

genus or species of the YAM fungi.

The stained samples, three per plot, were placed in

seperate counting dishes. Root and fungal intercepts for

every dish were counted. Each sample was then mixed and

new counts were made, which is referred to as a trial.

Three trials per sample were taken and then reported as an

average. At least 100 intercepts were counted per trial.

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Using this approach Giovanetti and Mosse (1980) achieved a

standard error of less than 4%.

2.3 RESULTS

The average % infections and standard deviations for

each turfgrass are shown in Table 3. Results of the

spring (May) sampling showed that there were considerable

differences in YAM infection rates between grasses.

Seaside creeping bentgrass had the highest % infection

<73%), where as Park Kentucky bluegrass had the lowest

(46%). The fall (October) sampling also showed

considerable differences in infection between grasses.

Ci ta tion perennial ryegrass wa s the highest (85 %) with

Bonnieblue Kentucky bluegrass the lowest (60%).

The % infection averaged over all turfgrasses varied

between sampling dates. There was a substantial increase

in YAM colonization over the course of a growing season.

The seasonal average of the two sampling dates showed

that Citation perennial ryegrass had the highest infection

and that Park and Bonnieblue Kentucky bluegrasses had

lowest.As can be seen in Table 4, there was little or no

difference in YAM infection between the fungicide treated

Kentucky bluegrass and the non-treated control. Over the

course of the growing season both the control and benomyl

treatments, had a substantial increase in YAM colonization

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TABLE 3

Mean 7. YAM infection levels of Field Surveyed turfgrassesduring May and October 1982

SAMPLING DATE

May 1982 Oct 1982

Mean S.D.* AverageInfection--------------

69 3 5878 7 6560 9 5868 6 64

TurfgrassKenyucky bluegrass

cv. Parkcv. Ram Icv. Bonniebluecv. Touchdown

Fine Fescuecv. Wintergreencv. Scaldis

Perennial Ryegrasscv. Citation

Creeping Bentgrasscv. Seaside

Average

Mean S.D.*------------7.46 1352 2555 1359 6

50 1557 7

69 6

73 12

57

7077

85

7472

102

7

12

6067

7774

*S.D.=Standard Deviation

compared to the triadimefon-treated turf. However,

averaged over the two sampling dates, there was little

difference between the systemically translocated

fungicide-treated areas and the non-treated control.

2.4 DISCUSSION

A major objective of this field survey was to firmly

establish the association between YAM fungi and

intensively managed cool season turfgrasses. As seen, in

Table 3, this was the case. One could conclude that YAM

fungi are indigenous in intensively managed turfgrasses

and infection occurred in at least 507. of the root system.

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TABLE 4

Mean 7. VAM infection levels of Kentucky bluegrass treatedwith fungicides

OctMay

SAMPLING DATE

1982 1982

Treatment

TriadimefonBenomylControl

Mean S.D.* Mean S.D.* Average--------------% Infection------------62 4 69 13 6661 4 74 8 6855 3 68 5 62

Average 59 4 70 3

*S.D.=Standard Deviation

These results are somewhat high in relation to the finding

of Rhodes and Lar sen (1981). They found the 7. infection

by VAM fungi on non-fungicide treated controls of creeping

bentgrass to be consistantly near 50%.

Another goal of this field survey was to examine the

effece of systemically translocated fungicides on VAM

colonization of the turfgrass root system. It was found

that the systemically translocated fungicides, tridimefon

and benomyl, did not substantially influence VAM

colonization of Kentucky bluegrass roots.

However, several researchers found that benomyl, even

at very low rates of application, not only depressed VAM

colonization but depressed VAM enhanced plant growth

(Jaoali and Domosch, 1975; Boatman et al., 1978; Nemec,

1979). It should be noted that the se researcher s used a

variety of plants not in the same family as turfgrasses.

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For insance Nemec (1979) quantified the VA mycorrhizal

infection of sour orange seedlings. He also applied the

fungicides (including benomyl) directly to the soil at the

same time VA mycorrhizal chlamycospores were being

mechanically mixed in the soil. In the ongoing longterm

fungicide study benomyl was applied as a foliar

application to an existing field of established Kentucky

bluegrass.

Rhodes and Larsen (1981), in a field study, found that

a single application of benomyl applied at a rate of 3 kg

ai/ha either in the spring or the fall reduced VA

mycorrhizal development on Pencross creeping bentgrass.

In their greenhouse study both triadimefon and benomyl,

when applied within 4 to 8 weeks after seeding,

dramatically reduced VA mycorrhizal development. However,

when either fungicide was applied 16 to 20 weeks after

seeding, VA mycorrhizal development was uneffected. This

would suggest that once the root system was colonized by

VA mycorrhiza, fungicides would not kill the existing VAM

infection. It is possible that the VA mycorrhiza

colonized the roots of the Kentucky bluegrass sod prior to

transplanting, or after transplant but before fungicides

were appl ied. This would explain the lack of fungicide

suppression of VA mycorrhizal development noted on the

longterm fungicide treated plots. However, a more

plausible explanation may exist in regard to the physical

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depth of sampling in this field survey. Both fungicides

were surface applied. However, benomyl, being very

insoluble in water, would concentrate mostly in the upper

soil surface (Smiley, 1984) and therefore could only limit

YAM root infection in this upper region. Although benomyl

moves both acropetally and basipitally, it moves downward

very slowly and only short distances (Smiley, 1984).

Triademfon, on the other hand, moves only acropetally

(Smiley, 1984). These factors could result in little or

no effect of triadimefon and benomyl on YAM infection

since the results shown here were average infection over

the surface 15cm depth of roots.