U.S. Department of Agriculture

Forest Seruice Research Note S E- 283 October 1979

SYNTHESIS OF Z’ZSOLZTHUS ECTOMYCORRHIZAE ON PECAN

SEEDLINGS IN FUMIGATED SOIL

by Donald H. Marx IABSTRACT.--Curtis variety of pecan (Carya iknoensis) seedlings were grown for 8 months in

fumigated soil infested at sowing with mycelial inoculum of Pisolithus rincrorius. Pisolirhus ectomycorrhizae were formed on all inoculated seedlings and significantly improved their growth over

control seedlings. inoculated and control seedlings also formed ectomycorrhizae with naturally

occurring Thelephora terrestris which produced abundant fruit bodies on seedling stems; most were

formed on control seedlings. The results showed that the techniques developed to form Pisolithus

ectomycorrhizae on pine seedlings are also applicable to pecan seedlings except that inoculum

should be incorporated 20 cm deep rather than IO cm, which is used for pine.

Keywords: Regeneration of hardwoods, symbiotic association, nursery seedling production, Thelephora terrestris, Catya illinoensis.

Pecan (Curya illinoensis Wang. K. Koch) is a

widely planted shade and orchard tree in the

southern part of the United States and in other

parts of the world (Smith and others 1974). Pecan

cultivars are usually grafted onto seedling rootstocks in the nursery, but clonal rootstocks can

also be produced by using various rooting and

air-layering techniques (Pokomy and Sparks

1967; Sparks and Pokomy 1966). The planting

stock is usually grown for 2 or more years in

fumigated nursery soil, then transplanted to

orchards either as bareroot or root-balled seedlings. Even with good cultural practices, many

seedlings do not survive transplanting, and those

that survive often do not grow appreciably in

height until the second or third year. Although

slow growth after outplanting is common to most

hardwood trees (Russell 197I), the cause is unknown. There is reason to believe, however, that

the problem may be at least partially alleviated by

producing nursery seedlings that are heavily ectomycorrhizal with highly beneficial fungi.

Pecan trees in urban or orchard plantings

have abundant ectomycorrhizae. Woodroof

‘Marx is Plant Pathologist and Director, Institute for

Mycorrhizal Research and Development, Forestry Sciences

Laboratory, Athens, Ga. 30602.

(1933) described the ectomycorrhizae of pecan in

Georgia orchards and listed several species of

fungi associated with them. More recently in

Georgia, Marx and Bryan (1969) found that

Scleroderma bovista Fr. formed abundant ectomycorrhizae following soil application of various

fungicides and nematocides in treatment of rootdiseased pecan trees. The appearance of this

specific ectomycorrhizae was concurrent with the

disappearance of foliar disease symptoms on the

trees. Nothing is known of the significance of

ectomycorrhizae to the physiological activity of

roots of transplanted pecan seedlings.

Recently, techniques were developed to

tailor pine and oak seedlings with Pisolithus

tinctorius (Pers.) Coker and Couch ectomycorrhizae in fumigated soils of tree nurseries (Marx

and others 1976; Marx and Artman 1978; Marx

and others 1978; Marx 1979a, 1979b). Pine seedlings with abundant Pisolithus ectomycorrhizae

formed in the nursery survive and grow better

after outplanting on various reforestation sites in

the South than seedlings with ectomycorrhizae

formed by naturally occurring fungi in the nursery

(Marx 1978; Marx and others 1977; Berry and

Marx 1978). Pisolithus basidiocarps have been

observed in pecan orchards in Arizona (Marx

Southeastern Forest Experiment Station - Asheuille, North Carolina
1977) and southern California (Menge, personal

communication). These observations indicate

that P. tinctorius is probably ectomycorrhizal on

pecan.

The research reported here had two purposes: (1) to determine whether the techniques

developed to tailor pine and oak seedlings with

Pisolithus ectomycorrhizae in nurseries would

also be effective on pecan, and (2) to determine

the significance of soil infestation with mycelial

inoculum of P. tinctorius to growth and ectomycorrhizal development of pecan seedlings in an

experimental nursery. Since these tests were

successful, field trials on the significance of Pisoiithus ectomycorrhizae to survival and growth of

pecan seedlings should be undertaken.

MATERIALS AND METHODS

Ten wood-frame microplots, each 1.2 x 0.5

x 0.3 m deep, were placed 1 m apart on level

ground. The frames and surrounding soil were

fumigated in place. Each frame was filled with a

fumigated mixture (2: 1: 1 volume ratio) of forest

clay loam, sand, and pine bark. The soil mixture

had a pH of 4.9 and contained 8 p/m of available P

and 59, 190, and 32 p/m of exchangeable K, Ca,

and Mg. Total nitrogen was 180 p/m and the organic matter content was 1.9 percent.2 All fumigation was done with methyl bromide (MC-2,

Dow Chemical Co., Midland, Michigan) at

recommended rates under clear polyethylene.

Vegetative inoculum of P. tinctorius (isolate

138) was grown for 3 months in a nutrient culture

of vermiculite and peat moss and processed as

previously described (Marx and Bryan 1975).

Leached inoculum (3 liters/m2 of soil surface),

commercial 10-10-10 fertilizer (280 kg/ha) and

hydrated lime (1008 kg/ha) were broadcast over

the soil surface of each of five microplots and

mechanically mixed into the upper 10 cm of soil.

Autoclaved inoculum (control) and the fertilizer

and lime at the same rates were mixed similarly

into the soil of the five remaining microplots.

Stratified pecan nuts of the Curtis variety

were cracked to hasten germination and planted

10 cm apart in two 1.2-m-long rows (11 seeds per

row) per microplot. Fumigated pine straw mulch

was placed 2 cm deep over the soil in each microplot. The study was installed in April 1976. During

‘Soil analysis done by Carol G. Wells, U.S. Department

of Agriculture Forest Service, Southeastern Forest Experiment Station. Forestry Sciences Laboratory. Research Tri-

-ogle Park, N. C. 27709.

the 1976 growing season, seedlings were fertilized

once with 36 kg N/ha (as NHdN03) in June and

July and were irrigated at least twice weekly after

seed germination. Starting in July, all microplots

were examined weekly for fruit bodies of P.

tinctorius and other ectomycorrhizal fungi and

their incidence recorded.

In December 1976, the seedlings were vertically cut between rows and undercut 20 cm with a

shovel. Seedlings were removed by hand and

washed in water. Heights, root-collar diameters,

and top (without leaves) and root fresh weights

were recorded for each seedling. The percentage

of feeder roots that were ectomycorrhizal were

visually estimated without magnification (Marx

and Bryan 1975). Randomly selected roots were

free-hand sectioned, mounted in phloxinelactophenol and examined at 100x to confirm the

presence or absence of the Hartig net and to

measure the thickness of the fungus mantle. All

data were subjected to analysis of variance at P =

0.05.

RESULTS

Pisofithus formed ectomycorrhizae on all

pecan seedlings in inoculated plots. Most of these

ectomycorrhizae were on the upper half (10 cm) of

the root system. Seedlings with Pisolithus ectomycorrhizae had significantly greater heights,

root-collar diameters, and root and top fresh

weights than the control seedlings (table 1). The

ectomycorrhizae formed by P. tinctorius were

morphologically similar (elongated coralloid) to

those formed on pecan by Scleroderma bovista

(Marx and Bryan 1%9), but were yellowish brown

rather than white. Many yellow-brown hyphal

strands were attached to these ectomycorrhizae.

The hyphae of P. tinctorius forming the Hartig net

penetrated between at least two layers of cortex

cells: fungus mantles were from 22 to 56 p thick.

No fruit bodies of P. tinctorius were observed in

inoculated plots.

Noninoculated control seedlings also formed

abundant ectomycorrhizae, but with naturally

occurring fungi. Forty-one fruit bodies of Thelephora terrestris Ehrh. ex Fr. occurred on seedling

stems in the control plots and 19 were observed on

seedlings in inoculated plots. Due to its numerous

associations with ectomycorrhizae of trees (Marx

1978), T. terrestris was assumed to be the most

prevalent naturally occurring fungus forming

ectomycorrhizae on the pecan seedlings. These

Thelephora ectomycorrhizae were light brown
Table 1 .-Growth and ectomycorrhizal development of pecan seedlings (Curtis variety) after 8 months

in fumigated soil artificially infested with Pisolithus tinctorius (Pt). Each number is the mean of 17

to 2 1 seedlings from each of five plots per treatment.

Feeder roots

Root- Fresh weights ectomycorrhizal with:

Treatment Height collar -

diameter Top Root Total Pt Other Total

-______ cm ________ _______. - ____ g _______________ -___-----_ percen, ----___----

Pisolithus

tinctorius 32.8* 0.71* 5.2* 22.0* 27.2* 24* 35* f50*

Noninoculated 28.9 0.64 3.9 18.0 21.9 0 56 56

*Significantly different from noninoculated seedlings (P = 0.05).

and were primarily bifurcate without much

repeated branching. Few hyphal strands were

apparent on these roots. The hyphae of the Hartig

net usually penetrated between only one cortex

cell layer; fungus mantles varied in thickness from

25 to 32 I_L.

DISCUSSION

It is apparent that the techniques developed

for forming Pisolithus ectomycorrhizae on pine

seedlings are also applicable for pecan seedlings.

However, the location of most Pisolithus ectomycorrhizae on the upper half of the root system

of the pecan seedlings in this study suggests that

the inoculum should be incorporated deeper than

LITERATURE CITED

Berrv. C. R., and D. H. Marx

i978. Effects of Pisolithus tinctorius ectomycorrhizae on growth of loblolly and Virginia pines

in the Tennessee Copper Basin. U.S. Dep.

Agric. For. Serv., Res. Note SE-264, 6 p.

Southeast. For. Exp. Stn., Asheville, N. C.

Marx, D. H.

1977.

Marx, D. H.

1978.

Marx, D. H.

1979a.

Tree host range and world distribution of the

ectomycorrhizal fungus Pisolithus tinctorius.

Can. J. Microbial. 23:217-223.

Ectomycorrhizal fungus inoculations: A tool

for improving forestation practices. IFS Workshop, Symp. Trop. Mycorrhizae Res., Aug.-

Sept. 1978. [Kumasi, Ghana].

Synthesis of ectomycorrhizae by different fungi

on northern red oak seedlings. U.S. Dep. Agric.

For. Serv., Res. Note SE-282, 8 p. Southeast.

For. Exp. Stn., Asheville, N. C.

the 10 cm used for pine. The majority of the feeder

roots of pecan was produced in the upper 20 cm of

soil; therefore, inoculum should be mixed to a

20-cm depth. Incorporating inoculum to this

depth should increase the amount of Pisolithus

ectomycorrhizae over that reported here. This

increase should benefit pecan seedlings since pine

seedlings with the greatest amount of Pisofithus

ectomycorrhizae at planting generally survive

and grow better than those with a smaller amount

of Pisolithus ectomycorrhizae (Marx and others

1977).

Since growth of pecan seedlings was stimulated by Pisolithus ectomycorrhizae in the

nursery, the potential of this fungus to improve

survival and growth of pecan in orchards should

be determined.

Marx, D. H.

1979b. Synthesis of Pisolithus ectomycorrhizae on

white oak seedlings in fumigated nursery soil.

U.S. Dep. Agric. For.Serv., Res. Note SE-280,

4p. Southeast. For. Exp. Stn., Asheville, N. C.

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1978. Growth and ectomycorrhizal development of

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terrestris in Virginia. U.S. Dep. Agric. For.

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*U.S. GOVERNMENT PRINTING OFFICE: 1979.640-190/4515. Region 4.