U.S. Department of Agriculture

Forest Service Research Note SE- 301 May 1981

PERFORMANCE OF CHOCTAWHATCHEE SAND PINE SEEDLINGS

INOCULATED WITH ECTOMYCORRHIZAL FUNGI

AND OUTPLANTED IN THE SANDHILLS OF NORTH FLORIDA

by John L. Ruehle’ and Raymond H. Brendemuehl’

ABSTRACT.-Studies were conducted on the effect of the ectomycorrhizal symbiont Pisolifhus

tincrorius (Pt) on nursery production and field performance of both container-grown and bare-root

Choctawhatchee sand pine (CSP). In two separate experiments, seedlings inoculated with Pt were

planted on prepared sites on the sandhills of north Florida. Inoculation with Pt improved the quality of

bare-root CSP in the nursery. Field survival and early growth were not affected by mycorrhizal treatment. The reasons for the failure of Pt inoculation to improve outplanted seedling performance during

these experiments are discussed.

Keywords: Pinus clausa var. immuginata, Pisolithus tinctorius, Thelephora terrestris, container stock.

reforestation, seedling quality.

It is generally recognized that if forest tree

species such as pinus are to grow normally, it is

essential that ectomycorrhizae develop on their

roots. However, not all mycorrhizal fungi are

equally beneficial or adapted to the wide range of

soil and climatic conditions supporting tree growth.

In his studies of coal spoil wastes in Pennsylvania,

Schramm (1966) noted that of the numerous species

of mycorrhizal fungi potentially available from

adjacent forested sites, only Pisolithus tinctorius

(Pt), Thekphora terrestris (Tt), and a few other

species were able to survive on these adverse sites

and form ectomycorrhizae. Marx and Bryan (1971)

have also noted this adaptation of Pt to such

adverse sites and reported that this symbiont

increases the tolerance of pines to high soil

temperatures.

The sandhills of north Florida without question

can be described as adverse sites. These soils are

droughty, infertile, and surface soil temperatures

become as high as 7 1°C when sites are exposed by

‘Plant Pathologist, Institute for Mycorrhizal Research and

Development, Forestry Sciences Laboratory, Athens, Georgia.

?3oil Scientist, Southeastern Forest Experiment Station,

Marianna, Florida.

site preparation. Pine seedlings planted in these

severe conditions are definitely subjected to periods

of stress. Of the pine taxa that have been tested, the

Choctawhatchee variety of sand pine (Pinus clausa

var. immuginata D.B. Ward), a species endemic to

this area, shows the greatest promise for sandhill

reforestation (Brendemuehk3 Burns 1973).

Despite the fact that Choctawhatchee sand pine

(CSP) is obviously well suited for sandhill reforestation, two deficiencies that may be related to

mycorrhizal development have been noted in the

early growth of this species. Seedling growth in the

nursery is often uneven; the larger seedlings have

excellent mycorrhizal development, while the

smaller ones usually have few mycorrhizal roots. In

addition, height growth of CSP seedlings during the

first 2 or 3 years in the field is often less than that of

Ocala (P. clausa var. clausa (Chapm.) Vasey) or

slash (P. elliottii Engelm.) pine, species that

frequently fail to attain merchantable size when

planted in the sandhills.

‘Brendemuehl, R. H. Options for management of sandhill

forest land. Southeastern Forest Experiment Station, Marianna,

Florida.[In process.]

Southeastern Forest Experiment Station - Asheuille, North Carolina
Reported here are the results of two experiments

which were intended to improve the development of

CSP planting stock and to increase early growth of

these seedlings when transplanted to sandhill sites.

One study was of bare-root nursery stock, the other

was of container-grown stock.

METHODS AND MATERIALS

Nursery Inoculation-Seedling Production

CSP seedlings intended for bare-root planting

were produced during 1975 in a nursery located on

the Chipola Experimental Forest, Calhoun County,

Florida. Soils in this nursery are infertile,

excessively drained sands of the Lakeland series. A

portion of the nursery that had never been in

production was used for this study. The area was

cleared of all vegetation, triple superphosphate was

broadcast at the rate of 268 kg/ ha, and the site was

tilled with a rototiller.

Early in April when soil temperatures were

above 16°C the nursery site was fumigated with

methyl bromide (MC-2) released under polyethylene at the rate of 1 kg/ 10mZ of nursery area. Four

days later the polyethylene was removed, the site

was tilled, and the nursery beds were formed. Four

days following this, the vegetative Pt inoculum,

prepared according to procedures described by

Marx and Bryan (1975) was applied by hand to

three nursery beds at the rate of 10.8 liter/ 10 m*.

The inoculum was mixed into the soil with a

rotovator to a depth of 10 to 15 cm. On three

additional beds, vermiculite without Pt inoculum

was applied and mixed with the soil to allow the

effects of the inoculum upon the seedlings to be

separated from any effects of vermiculite alone. A

third set of nursery beds used to produce the control

seedlings received no inoculum.

Seed obtained from natural CSP stands in west

Florida were sowed in rows 15 cm apart and at a rate

calculated to produce approximately 325 seedlings

per square meter of nursery bed. The nursery

fertilization schedule developed by Brendemuehl

and Mizell(1978) for production of CSP seedlings

was followed.

Pt basidiocarps were first detected in the

inoculated beds in early September and were

removed at weekly inspection periods during the

balance of the year to prevent their spread to

adjacent control beds.

In January 1976, the seedlings were lifted and 90

seedlings per treatment were weighed (top weight

and root weight) and the roots were evaluated for

mycorrhizal development. Seedlings used for field

planting were graded for size and mycorrhizal

development. The seedlings lifted from Pt beds had

a very low percentage of their roots colonized with

Pt. Many of the Pt ectomycorrhizae were developed

on roots deeper in the soil and were lost during

lifting. Therefore, plantable seedlings used to

evaluate this treatment in the field plots had only 5

to 10 percent Pt ectomycorrhizae and approximately 25 percent other ectomycorrhizae (mostly

Tt). Also, seedlings with less than 10 cm of stem

supporting live needles or seedlings damaged in lifting were considered culls and were not planted. The

seedlings were machine-planted the same day at an

approximate spacing of 1.8 by 3 meters. About 125

seedlings were planted on the individual treatment

plots, each measuring 21 by 30 meters. All plots

were separated by a 3-meter isolation strip.

The five sites on the Chipola Experimental

Forest selected for the field plantings were typical of

the sandhills and had supported scrub oak and wire

grass before being double chopped in preparation

for planting. All five sites had excessively drained

sand soil profiles to a depth of at least 3 meters.

Laboratory analyses of samples taken from the

surface horizon (0 to 20 cm) of each site showed that

there was little variation among them. Average

values for the soil characteristics analyzed were: pH,

5.3; organic matter, very low; CaO, 84 kg/ ha; MgO,

15 kg/ha; PzO~, 6 kg/ ha; and KzO, 17 kg/ha.

One and two growing seasons after planting

(November 1976 and 1977), survival counts were

made and surviving trees in all plots were measured

for root-collar diameter and height. After determining the average height and root-collar diameter of

all seedlings in a plot, the smallest and largest

seedlings were harvested. A third seedling was also

chosen so that the average height and root-collar

diameter of the three seedlings approximated

(within 10 percent) the averages for that plot. The

top fresh weight of these seedlings was determined

onsite immediately after cutting the stems at ground

level. Another three seedlings were randomly

selected from each plot for mycorrhizal assessment

and dug up with a shovel. After carefully removing

most of the soil, the roots were visually assessed for

mycorrhizal development. All growth data were

subjected to an analysis of variance.
Container Inoculation-Seedling Production

Container-grown CSP seedlings were produced

in a greenhouse in Athens, Georgia. The seed were

from the same seed lot used the previous year in the

nursery inoculation study.

Inocula of Pt, isolate 138, and Tt, isolate 201,

were produced in peat moss-vermiculite nutrient

medium and processed as reported by Marx and

Bryan (1975). Mycorrhizal inoculum was mixed in a

1:6 v/v ratio with milled pine bark previously fumigated with methyl bromide. Autoclaved Pt

inoculum was mixed with fumigated pine bark at

the same rate for the control seedlings. Spencier

LeMaire Rootrainers@ (Hillson model, 150 cm3/

cavity) were filled with the growing medium mixed

with inoculum.

In May 1976, all containers were placed on

greenhouse benches and sowed with several seed per

cavity. After germination, seedlings were thinned to

one per cavity. Seedlings were watered as needed,

and fertilized until August with a commercially

available water-soluble NPK fertilizer (23: 19: 17)

and chelated iron as previously described (Ruehle

and Marx 1977). In August, all seedlings were

transported to a greenhouse at the Chipola Experimental Forest.

Before outplanting, 10 seedlings were randomly

selected from each of the three groups of seedlings.

Their roots were carefully separated from the

potting medium and visually assessed for

mycorrhizal development. Pt seedlings averaged 64

percent Pt and 8 percent naturally occurring Tt, Tt

seedlings averaged 68 percent Tt, and controls

averaged 40 percent naturally occurring Tt. All

seedlings were of uniform size regardless of treatment; they averaged 19 cm in height and 2 mm in

root-collar diameter.

Three planting sites-one on the Chipola

Experimental Forest and two on private land in

Washington County, Florida-were selected and

double shopped with a duplex brush cutter 3 to 4

months before planting. The sites were similar, with

infertile, excessively drained sandy soils (Lakeland

or Kershaw soil series) which supported scrub oak

and wire grass before being prepared for planting to

pines. The soil profile at each site was sand to a

minimum depth of 3 meters.

Laboratory analyses of soil samples collected

from the 0- to 20-cm surface of the soil profile at

each site indicated that the study areas were quite

similar in terms of chemical characteristics,

although site 1 was somewhat more fertile (table 1).

In December, seedlings were handplanted at a

spacing of 2.4 by 2.7 meters into 25-tree plots, each

comprised of five rows of five seedlings. To minimize contamination, all plots were separated by 3-

meter unplanted strips. Treatments were assigned to

plots in a randomized block design of five

replications.

In November 1978, 2 years after planting, the

survival, height, rootcollar diameter, selection and

harvesting, top fresh weight, and mycorrhizal

assessment of outplanted container-grown

seedlings were determined by the same procedures

as for the bare-root outplantings.

Table 1 .-Chemical properties of soils at outplanting sites of the container-grown

seedling study

I

Site Organic Soil property

matter PH PZOS MgG KzO CaO

__________________kglha________________

1 (Forests & Lakes) Very low 5.3 3.4 30.2 32.5 313.6

2 (Hunt) Low 5.1 3.4 14.6 21.3 100.8

3 (Chipola) Very low 5.2 3.4 14.6 16.8 89.6
RESULTS AND DISCUSSION

Field survival of both bare-root stock (nursery

inoculation) and container-grown stock (container

inoculation) was greater than 90 percent for all

plantings and did not vary by mycorrhizal treatment (table 2.) Treatments had no effect on survival. These results were not unexpected since

similar results had been obtained in experimental

and operational plantings of CSP seedlings which

were correctly handled and planted.

Average height, diameter, and aboveground

biomass of seedlings did not vary by mycorrhizal

treatment (tables 3 and 4). In the bare-root outplanting, this may have been due to the low percentage of Pt ectomycorrhizal development (5 to 10

percent).

In a container-stock outplanting test with shortleaf pine (Ruehle and others, In press), seedlings

colonized with Pt performed no better than control

seedlings. Excavation of samples of the container

stock after 2 years showed that Pt failed to spread to

the new roots which had emerged from the root

plugs. The root configuration was markedly different on container-grown seedlings than bare-root

seedlings and probably accounted, to some degree,

for the lack of spread of Pt and the consequent lack

of growth response with the container stock.

Similarly, after excavating sample seedlings of

inoculated CSP container stock 2 years after planting, we found that Pt again failed in most cases to

spread to the new roots that had emerged from the

root plug:

Table 2.-Effect of mycorrhizal treatment on second-year survival of CSP seedlings

Seedling type and

planting site

Nursery

Chipola

Container

Site 1

(Forests & Lakes)

Container

Site 2 (Hunt)

Container

Site 3 (Chipola)

Treatment’

Control Pt Tt Vermiculite

___ _ _ ____________Percent ________-____ ____

97.2 97.6 -_ 99.4

99.2 97.6 99.2 __

96.8 96.8 95.2 __

95.2 92.0 92.0 _ _

“Control-no inoculum, Pt-F’isolithus rincrorius inoculum, Tt-Thelephora rerresrris inoculum.

vermiculite-vermiculite carrier applied with no inoculum.
Table 3.-Effect of mycorrhizal treatment on second-year growth of CSP seedlings

I Treatment’

Planting site Control Pt Tt Vermiculite

Height Diameter Height Diameter Height Diameter Height Diameter

Nursery

Chipola

cm m m cm m m cm m m cm mm

64.0 -- 61.6 __ -- __ 64.6 --

Container

Site 1 (Forests & Lakes) 14.3 14.5 18.9 15.9 19.1 16.3 -- --

Container

Site 2 (Hunt) 49.7 8.4 50.3 8.5 44.8 7.2 -- --

Container

Site 3 (Chipola) 66.1 13.0 69.8 13.5 68.6 13.0 -- --

“Control-no inoculum, Pt-Pisolithus tinctorius inoculum, Tt-Thelephora terrestris inoculum. vermiculite-vermiculite

carrier applied with no inoculum.

Table 4.-Aboveground biomass of CSP seedlings. Means of five replicates

Treatment

Number of

seedlings

per plot

Seedling

stem volumea PVIb Top fresh PWI”

(cm3) (%103) weight (9103)

SITE 1

Pt 24.4

Tt 24.8

X 24.8

Pt 24.2

Tt 23.8

X 24.2

Pt 23.0 133.3 3.07 304.8 7.05

Tt 23.0 124.6 2.87 331.7 7.35

X 23.8 120.4 2.83 328.7 7.46

205.9 5.00

219.4 5.42

161.0 4.00

SITE 2

39.6 .96

23.5 .58

39.7 .93

SITE 3

432.7 10.56

494.0 12.20

409.4 10.17

117.1 4.30

143.8 3.44

175.3 4.21

“Seedling voIdme (cm)) = (rootcollar diameter)l X height.

bPVI is plot volume index (cm’) = mean seedling volume X number surviving seedlings.

‘PWI is plot weight index (g) = mean seedling top fresh weight X number surviving seedlings.
Planting

site

Proportion of inoculated Average percent of Ft

plugs still colonized colonization on roots which

with Pt had emerged from the plugs

1 1 3 of 1 5

2 11 of 1 5

3 13 of 15

In the CSP container-stock outplanting, as in

the shortleaf container-stock study (Ruehle and

others, In press), we found extensive colonization

by indigenous symbionts of roots that had emerged

from the plugs. We concluded that this natural

inoculation partly accounted for the low percentages of Pt colonization listed in the tabulation

above. Seedlings sampled from the other treatments

also had abundant ectomycorrhizae formed by

indigenous symbionts both in plugs or roots that

emerged from the plugs. Such early colonization

has been previously reported by Tainter and

Walsted (1977) for container-grown loblolly pine on

Coastal Plain sites.

One factor that could partly account for lack of

response to Pt inoculation in our bare-root seedling

study was the low percentage colonization by Pt (5

to 10 percent) on seedlings after lifting. To obtain

significant growth response of bare-root pine

seedlings on reforestation sites, a threshold level of

at least half of all ectomycorrhizae on seedlings at

planting must be formed by Pt (Marx and others

1977; Ruehle and others, In press; Kais and others4).

In the nursery phase of this study, the seedlings

grown in soil with Pt were much more uniform in

size than those grown in the uninoculated beds. The

high degree of seedling uniformity as a result of Pt

inoculation should help to make CSP seedling production more efficient. Unfortunately, too many Pt

ectomycorrhizae were lost during the lifting and

grading of these seedlings. This study, then, indicates that inoculation with Pt can improve nursery

grade of bare-root stock and suggests that more

research into the inoculation, increased colonization, and handling of CSP seedlings with Pt ectomycorrhizae is warranted.

23

10

20

LITERATURE CITED

Brendemuehl, R. H., and L. Mizell.

1978. Nursery practices for Choctawhatchee sand pine.

Tree Plant. Notes 29:8-l 1.

Burns, R. M.

1973. Comparative growth of planted pines in the sandhills of Florida, Georgia, and South Carolina. Sand

Pine Symp. Proc., USDA For. Serv. Gen. Tech. Rep.

SE-2, p. 124-134. Southeast. For. Exp. Stn., Asheville, NC.

Marx, D. H., and W. C. Bryan

1971. Influence of ectomycorrhizae on survivalandgrowth

of aseptic seedlings of loblolly pine at high temperatures. For. Sci. 19:3741.

Marx, D. H., and W. C. Bryan.

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

the fungal symbiont Pisolirhus finctorius. For. Sci.

21:245-254.

Marx, D. H., W. C. Bryan, and C. E. Cordell.

1977. Survival and growth of pine seedlings with PisoMn4.5 finctorius after two years on reforestation

sites in North Carolina and Florida. For. Sci.

231363-373.

Ruehle, J. L., and D. H. Marx.

1977. Developing ectomycorrhizae on containerized pine

seedlings. USDA For. Serv., Res. Note SE-242. 8 p.

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

Ruehle, J. L., D. H. Marx, J. P. Barnett. and W. H. Pawuk.

In press. Survival and growth of container-grown and bareroot shortleaf pine seedlings with Pisolithus and

Dzelephoru ectomycorrhizae. South. J. Appl. For.

Schramm, J. R.

1966. Plant colonization studies on black wastes from

anthracite mining in Pennsylvania. Trans. Am.

Phila. Sot. 56:1-194.

Tainter, F. H., and J. D. Walsted.

1977. Colonization of outplanted loblolly pines by native

ectomycorrhizal fungi. For. Sci. 23:77-79.

‘Kais, A. G., G. A. Snow, and D. H. Marx. The effects of

benomyl and Pisolithus tinctorius ectomycorrhizae on survival

and growth of longleaf pine seedlings. Southern Forest

Experiment Station, Gulfport, Mississippi. [In process.]
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‘??s wldlife--if they are not handled or applied properly Use all pesticides selectively and carefully. Follow

_-.- recommended practices for the disposal of surplus pesticides and pesticide containers. I

7
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U.S. ooverr*nnt Prlntln. Of‘iat *VW- ,35-056,4003 R.iOll No. a-11