U.S. Department

forest Seroice

of Agriculture

Research Note S E - 297

ECTOMYCORRHIZAL COLONIZATION OF

Jury 1980

CONTAINER-GROWN NORTHERN RED OAK

AS AFFECTED BY FERTILITY

by John L. Ruehlel

ABSTRACT.-The effects of different fertility regimes on Pisolithus tinctorius ectomycorrhizal

development on northern red oak grown in containers was tested in the greenhouse. Artificial

infestation of a milled pine bark-sewage sludge growing medium produced the best ectomycorrhizal

development (63 percent) when a nutrient solution with 100 pg/ml N and 20 pg/ml P was used every 3

weeks. A commercially available nutrient solution (NP-500 &ml) produced the largest seedlings,

those rated suitable for planting, but caused a marked reduction in ectomycorrhizal development (Pt

= 20 percent).

The results indicate that the production of large and vigorous northern red oak seedlings in

containers with abundant Pisolithus ectomyconizae is strongly dependent on the levels of available

nutrients in the growing medium.

Keywords: Pisolithus tinctorius, Quercus rubra.

Northern red oak (Quercus ruhra L.) grows

best in upland coves and middle and lower slopes

having a fine-textured soil and topography favoring a high water table (Core 197 1). Intense competition makes it difficult to reproduce red oak

stands on the better sites. There is a high mortality

of newly established oak seedlings in this region

(Clark and Watt 1971). Although reducing competition helps artificial oak regeneration, mowing

and use of herbicides are not feasible in many

areas in the uplands within the range of northern

red oak (Russell 1971).

Conventional nursery stock has not demonstrated the ability to grow fast enough to compete

with woody vegetation on forest sites (Johnson

1979). The advent of containerization in recent

years offers a possible means of improving early

seedling growth of oaks. Initial trials by Johnson

(1974) and Wendel(l979) showed growth rates of

containerized northern red oak to be too slow to

‘Ruehle is Plant Pathologist, Institute for Mycorrhizal

Research and Beveiopment, USDA Forest Service, Forestry

Sciences Laboratory, Athens, Georgia30602.

be competitive. Johnson speculated that a larger

container and a longer propagation period offered

the best opportunities for improving the growth of

outplanted tublings.

The use of ectomycorrhizal fungi ecologically adapted to the planting site in container operations may also improve survival and early

seedling growth (Ruehle and Marx 1979). These

symbiotic fungi assist the plant in water and mineral uptake by increasing the effective absorbing

area of the feeder roots. Good nonmycorrhizal

oak seedlings can be grown in containers in the

greenhouse because conditions are controlled to

minimize stress of all kinds (Tinus and McDonald

1979). However, such seedlings often fail to survive or grow well after planting, especially during

a period of environmental stress. Pine seedlings

colonized with Pisolithus tinctorius survive and

grow better on routine reforestation sites than

seedlings colonized with Thelephora terrestris or

other fungi (Marx and others 1977a). Since this

symbiont also colonizes oaks, it would be a good

candidate for tailoring the roots of oaks before

outplanting (Marx 1979). Previous work (Ruehle.

unpublished data) has shown that northern red

Southeastern Forest Experiment Station - Asheuille, North Carolina
oak became colonized with P. tinctorius when

inoculum was added to the growing medium.

However, the colonization was erratic and usually quite low (below 5 percent) in these tests

where trees received a commercially available

soluble fertilizer (NPK at 500 p/m each concentration) every 3 weeks. Before field trials can be

conducted to evaluate the performance of containerized seedlings colonized with specific ectomycorrhizae selected to adapt the seedlings to the

site, greenhouse experiments must be conducted

to determine the optimum growing medium, incculation techniques, and fertility levels needed to

produce acceptable plantable seedlings. Current

cultural practices for growing containerized oaks

are often not suitable for colonization of roots by

certain ectomycorrhizal fungi. Frequent watering

of seedlings in containers with poor drainage

(growing medium that is too fine often becomes

waterlogged) prevents feeder-root development.

Lack of feeder roots prevents adequate ectomycorrhizal development. Frequent application

of high concentrations of water-soluble fertilizer

also prevents mycorrhizal development by many

symbiotic fungi (Marx and others 1977b).

-The study was conducted to determine the

best fertility regime for developing P. tinctorius

ectomycorrhizae on northern red oak grown in

containers.

METHODS AND MATERIALS

Milled pine bark used as a growing medium

for containerized southern pine seedlings promotes extensive secondary-root development

and a high percentage of ectomycorrhizae

(Ruehle and Marx 1977), and was used in this

study. Processed sewage sludge was added to

milled pine bark because it permits less frequent

application of soluble fertilizer. Sewage sludge

used in this study was obtained from two primary

plants in Athens, Georgia, screened and dried 48

hours at 80°C. The nutrient content and organic

matter of this sludge was discussed by Berry and

Marx (1976). The milled pine bark was fumigated

with methyl bromide and then mixed 4: 1 v/v with

the processed sewage sludge to create the basic

growing medium for this study. Inoculum of P.

tinctorius (isolate 185) prepared according to

standard procedures (Marx and Bryan 1975) was

dried at room temperature to approximately 20

percent moisture, and mixed with the growing

medium at a 1: 15 v/v ratio.

Fifty plywood boxes were constructed with

screen bottoms. Each was large enough to hold

five Super-45 Spencer-LeMaire Rootrainers@

(Spencer-LeMaire Industries Ltd., Edmonton,

Alberta, Canada). Each Rootrainer had three

cavities; each cavity was 575 cm”. After each box

was filled with Rootrainers (15 cavities/box), 200

cm” of growing medium was added to each cavity.

The growing medium mixed with inoculum was

then added (350 cm”) to each cavity to fill it to

within 3 cm of the top.

A mixed lot of northern red oak seed collected in western North Carolina was Clorox@

treated, sorted by flotation, and stored in plastic

bags in a refrigerator for 2 months. The seed was

planted (one per cavity) in March 1978. Additional seed were planted in trays to provide transplants for cavities lacking seedlings. Three weeks

after seeding, those cavities lacking seedlings received transplants from the trays.

A nutrient solution modified from one employed by Marx and others (1977b) and a Rapidgro@ solution (control) was used in this test to

provide various levels of N and P (table 1). Treatments assigned to each group of 10 boxes are

listed in table 2.

Table 1 .-Modified nutrient solutions for fertility

study on northern red oak grown in containers

Concentration

N

P

K

Ca

Mg

Fe

MO

c u

Bo

Zn

Mn

tLglml

NH,NO, 12.5,25,50, or 100

Na*HPO, 5or20

KC1 40

CaCl* 40

MgS0,.7H,O 20

Chelated Fe 10% 5.5

NaMo04.7H,0 .OOl

CuS0,*5H20 .006

H3B03 .09

ZnS0,.7H20 .1

MnCl,*4H,O .7

The solutions were added to saturation (= 50

ml) to each cavity at 2,5,8, 11, 14, and 17 weeks

after germination of seedlings. The treatments

were completely randomized on the greenhouse

bench with each treatment replicated 10 times;

each replicate was a box of 15 seedlings.

After 20 weeks, five randomly selected seedlings were removed from each box and the growing medium was gently washed from the roots.
Table 2.-Treatments for fertility study on

northern red oak grown in containers, by

nutrient concentration

Treatment

IN FT K

. . _. . . . . . . . . . . . . . .pglml . . . . . . . . . . . . . . .

MNS’

1 12.5 (N,) 5 (P,) 40

2 25 (N2) 5 (PI) 40

3 50 (NJ 20 (PJ 40

4 100 (Ns) 20 (PJ) 40

Rapidgro@ 500 500 500

‘MN.%-Modified nutrient solution with various levels of

NPK.

Height, root-collar diameter, and fresh top and

root weight were determined for each seedling.

The roots were visually assessed for degree of

ectomycorrhizal development.2

Tops and roots of 10 seedlings per replicate

wereovendried at 80°C for 60 hours and weighed.

Leaves were removed and analyzed for total N by

the Kjeldahl method, and for the other elements

by dry-ash methods (Wells and others 1973).”

RESULTS

Seedlings receiving Rapidgro@’ produced

greater height and root-collar diameter than those

receiving the modified nutrient solutions (fig. 1).

There was no difference in the two parameters

among seedlings receiving the modified nutrient

solutions. Top dry weight followed the same pattern between treatments with seedlings receiving

the Rapidgro@ solution producing four times more

top dry weight than those receiving other nutrient

solutions (fig. 2). Seedlings fertilized with the

Rapidgro@ solution also had the greatest amount

of root dry weight. Seedlings receiving NgP4

Bt_ttrient solution had significantly greater root

weight than those receiving treatment 1 Or

Rapidgro@.

‘Ectomycorrhizal assessments were made by Donald H.

Marx, Institute for Mycorrhizal Research and Development,

Forestry Sciences Laboratory, Athens, Georgia.

:lAnalyses of foliar samples were performed by Carol G.

Wells, USDA Forest Service, Forestry Sciences Laboratory.

Research Triangle Park. N.C.

50

40

2 i

0 SHOOT HEIGHT

0 ROOT-COLLAR DIAMETER

DIAYETERlmm) @ @ @

FERTILITY REGIME

Figure 1 .-Shoot height and root-collar diameter of northern

red oak seedlings at 15 weeks under five fertility regimes.

Bars or circles bearing the same letter are not significantly

different (P = 0.05).

6- 8

Bk 0.

$ 2-

6 4 I---- 0 ab ab b

8

6 8

I

NIPI %P, NeP4 NsC Rap

FERTILITY REGIME

Figure 2.-Shoot and root dry weight under five fertility

regimes. Bars bearing the same letter are not significantly

different (P = 0.05).

The percentage of N in the foliage related

well with the increasing amounts of N in the

nutrient solutions (table 3). The level of N in the

foliage of seedlings receiving Rapidgro@ was quite

high, probably approaching the luxuriant consumption range. The level of foliar P was slightly

affected by fertility regime but levels of K, Ca,

Mg, and Fe were not.

The highest percentage of ectomycorrhizae

developed on seedlings in N4P4 and NsP4 treatments (table 4); significantly less formed on seedlings receiving the highest levels of N and P

(Rapidgro@). These findings agree with those of

3
Table 3.-Foliar analysis of 15week-old northern red oak grown under five fertility

regimes in containers,’ by nutrient

Treatment2 N P K Ca Mg Fe

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Percent . . . . . . . . . . . . . . . . . . . . . .

NIPI 0.89d3 0.46b l.l@ 0.93a 0.23a O.Ola

N2P1 .92cd .47b 1.25a .91a .22a .Ola

N4P4 1.03c .53a 1.23a 1.02a .25a .Ola

W’4 1.17b .44b 1.12a l.oOa .22a .Ola

Rapidgro@ 2.02a .42b l.oOa .92a .25a .Ola

‘Values are means of 10 replicates; each replicate was a composite of leaves from 10 seedlings.

Toncentrations of nitrogen as pg/ml - Nl = 12.5, N2 = 25, N4 = 50, Ng = 100; phosphorus as

pg/ml - Pl = 5, P4 = 20; Rapidgro” - NPK = 500 pg/ml each.

3Means within columns not followed by the same letter are significantly different (P = 0.05).

Table 4.-Percentage of seedlings with ectomycorrhizae and degree of ectomycorrhizal

development by Pisolithus tinctorius (F’t)l

NPl

N2P1

N4P4

N8P4

Rapidgro@

. . . . . . . . Percent . . . . . . . .

99 37.0b3

100 40.7b

100 58.6~

100 63.5~

89 20.7a

‘Values are means of five seedlings from each of IO

replicates.

‘Concentrations of nitrogen as pglml - N 1 = 12.5. N2 =

25. N4 = 50. N8 = 100: phosphorus as fig/ml - Pi = 5. P4 =

20: RapidgroB - NPK = 500 @g/ml each.

:‘Means not followed by the same letter are significantly

different (P = 0.05).

Marx and others (1977b) working with P. tinctorius on pine seedlings. Naturally occurring

ectomycorrhizae (mostly Thelephora terrestris)

formed approximately 5 to 10 percent of the total

mycorrhizal development on all seedlings.

CONCLUSIONS

Northern red oak seedlings will grow to

plantable size in containers filled with 730 cm3 of

pine bark-sludge growing medium in 3% to 4

months when fertilized with Rapidgro@ every 3

weeks. However, ectomycorrhizal development

by Pisolithus tinctorius is less than satisfactory

under this fertility regime. More Pisolithus ectomycorrhizae developed with a balanced nutrient

solution with lower concentrations of N and P.

Treatments 3 (N4P4) and 4 (NsP4) produced seedlings with excellent ectomycorrhizal development under the conditions of this study. Unfortunately, seedlings under these fertility regimes

were still slightly chlorotic (2.5 GY/7.5/5/9) compared to the dark-green foliage on plants receiving

Rapidgro@ (7.5 GY/4.5/5).’ Seedlings under treatment 3 or 4 also lacked appropriate stem height

and root collar diameter. Tinus and McDonald

(1979) state that container-grown bur oak

(Quercus macrocarpa Michx.) should be 20 to 25

cm tall before outplanting. Northern red oak

probably should be at least this tall before planting, and possibly up to 35 to 40 cm, the height of

seedlings receiving Rapidgro@.

Work is being continued to find the fertility

regime that will produce northern red oak in containers having the robust tops and roots of seedlings receiving abundant N and P and the P. tinctorius colonization of those receiving less

amounts of N and P. The use of sewage sludge

also contributes approximately 10 to 20 pg/ml of

N and P to the fertility regime when used in the

growing medium. A chemical analysis of the

growing medium before and after the addition of

nutrient solutions needs to be done to define the

actual levels of available N and P.

4Foliage-color rating with Munsell color charts for plant

tissue (hue/value/chroma).

4
LITERATURE CITED

Berry, Charles R., and Donald H. Marx.

1976. Sewage sludge and Pisolithus trnctorius ectomycorrhizae: Their effect on growth of pine seedlings. For. Sci. 22:351-358.

Clark, F. Bryan, and Richard F. Watt.

1971. Silvicultural methods for regenerating oaks. Proc.

Oak Symp., p. 37-43. [Morgantown, W. Va.,

August 1971.1 Northeast. For. Exp. Stn., Upper

Darby, Pa.

Core, Earl L.

1971. Silvical characteristics of the five upland oaks.

Proc. Oak Symp., p. 19-22. [Morgantown,

W. Va., August 1971.1 Northeast. For. Exp. Stn.,

Upper Darby, Pa.

Johnson, Paul S.

1974. Containerization of oak seedlings for the oakhickory region: A progress report. Proc. North

Am. Containerized For. Tree Seedling Symp.,

p. 197-199. [Denver, Cola., August 1974.1 Great

Plains Agric. Count. Publ. 68.

Johnson, R. L.

1979. Adequate oak regeneration: A problem without a

solution. Proc. 7th Annu. Hardwood Symp.,

Hardwood Res. Count., p. 59-65. [Cashiers,

N.C., May 1979.1 USDA For. Serv., Southeast.

Area, State and Priv. For., Atlanta, Ga.

Marx, Donald H.

1979. Synthesis of ectomycorrhizae by different fungi

on northern red oak. USDA For. Serv., Res. Note

SE-282,8 p. Southeast. For. Exp. Stn., Asheville,

N.C.

Marx, Donald H., and William C. Bryan.

1975. Growth and ectomycorrhizal development of lob1011~ pine seedlings in fumigated soil infested with

the fungal symbiont Pisolithus tinctorius. For.

Sci. 21:245-254.

Marx, Donald H., William C. Bryan, and Charles E. Cordell.

1977a. Survival and growth of pine seedlings with Pisolithus ectomycorrhizae after two years on reforestation sites in North Carolina and Florida. For.

Sci. 23:363-373.

Marx, D. H., A. B. Hatch, and Joseph F. Mendicino.

1977b. High soil fertility decreases sucrose content and

susceptibility of loblolly pine roots to ectomycorrhizal infection by Pisolirhus tinctorius. Can. J.

Bot. 55: 1569-1574.

Ruehle, John L., and Donald H. Marx.

1977. Developing ectomycorrhizae on containerized

pine seedlings. USDA For. Serv., Res. Note SE242, 8 p. Southeast. For. Exp. Stn., Asheville,

N.C.

Ruehle, John L., and Donald H. Marx.

1979. Fiber, food, fuel, and fungal symbionts. Science

206:419422.

Russell, Thomas E.

1971. Seeding and planting upland oaks. Proc. Oak

Symp., p. 49-54. [Morgantown, W. Va., August

1971.1 Northeast. For. Exp. Stn., Upper Darby,

Pa.

Tinus, Richard W., and Stephen E. McDonald.

1979. How to grow tree seedlings in containers in greenhouses. USDA For. Serv., Gen. Tech. Rep. RM60, 256 p. Rocky Mt. For. and Range Exp. Stn.,

Fort Collins, Colo.

Wells, Carol G., D. M. Crutchfield, N. M. Berenyi, and

Charles B. Davey.

1973. Soil and fohar guidelines for phosphorus fertilization of loblolly pine. USDA For. Serv., Res. Note

SE-I 10, 15 p. Southeast. For. Exp. Stn., Asheville,

N.C.

Wendel, George W.

1979. Growth and survival of planted northern red oak

seedlings in West Virginia. USDA For. Serv.,

Tree Plant. Notes 30: 16-19.

The use of trade, firm, or corporation names

in this publication is for the information and

convenience of the reader. Such use does not

constitute an official endorsement or approval by the U . S . Department of Agriculture

or the Forest Service of any product or

service to the exclusion of others that may be

suitable.

This publication reports research involving pesticides. It does not contain recommendations for their use,

nor does it imply that the uses discussed here have been registered. All uses of pesticides must be registered by appropriate State and/or Federal agencies before they can be recommended.

CAUTION: Pesticides can be injurious to humans, domestic animals, desirable plants, and fish or other

&EL%$ wildlife--if they are not handled or applied properly. Use all pesticides selectively and carefully. Follow

_-.- recommended nractices for’ the disposal of surplus pesticides and pesticide containers.

5


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