k United statesq 

Department of 

Agriculture 

Forest Service ' 

Southeastern Forest 

3 Experiment Station 

Research Paper 

t"S~-258 

Ecology of the Pinewood 

Nematode in Southern 

Pine Chip Piles 

L. David Dwinell 
Ecology of the Pinewood Nematode 

in Southern Pine Chip Piles 

L. David Dwinetl 

Research Plant Pathologist 

Forestry Sciences Laboratory 

Athens, Georgia 

ABSTRACT 

In wood-chip piles of southern pine species, the pincwood nematode was found mainly in 

fresh chips and chips located in the outer shells of piles. Chips in the interior of 0 

piles did not harbor the nematode if the temperature reached 60 C, caused by wood-cell 

respiration. In naturally infested or nematode-enhanced chips, the optimum temperature 

range for reproduction of the pinewood nematode was 35 to It0 OC. Samples of truck 

shipments of chips varied in response to incubation at 1&0 OC. Mematode populations 0 

declined rapidly at temperatures above 45 C. There was no marked change in population 0 

density for 21 days at temperatures of 0 to 10 C. The nematode did not survive in 0 

chips at -20 C for more than 3 days. In chips that had been autoclaved and infested 

with the bluestain fungus (Ceratocystis sp.), the optimum temperature for reproduction of 0 

the nematode was 35 C. Significantly more nematodes were recovered from chips colonized by bluestain than those not colonized. Wood moisture had a marked impact on the 

population density of the nematode in chips. The pinewood nematode was intolerant of an 

anaerobic environment at 38 OC. Pinewood nematodes extracted from southern pine chips 

were pathogenic on 2-year-old slash pine seedlings in the greenhouse. 

$ Keywords: Bursaphelenchus xylophilus, temperature, bluestain, pine wilt, slash pine, 

loblolly pine, pathogen. 

The pinewood nematode, 

Bursaphelenchus xylophilus (Steiner & 

Buhrer) Nickle (= B. lignicolus Mamiya & 

Kiyohara), and pine sawyers, Monochamus 

spp., are the primary causal agents of 

the pine wilt disease. Although the 

impact of the pine wilt complex on 

forests in Japan has been well documented (Mamiya 1983, 1984), the role of 

the disease in the United States is 

still being defined (Wingfield and 

others 1982). In most cases, pine wilt 

is associated with exposure to environmental stress (Mamiya 1983, 1984; 

Wingfield and others 1982). The disease 

was not considered a problem in southern 

pines until the pinewood nematode was 

discovered in dying pines in several 

seed orchards (Dwinell and 

Barrows-Broaddus 1983). 

All 43 species of Bursaphelenchus 

described in the literature have a phoretic relationship with insects, especially bark beetles and wood borers, and 

all are mycophagous (Massey 1974; Hiihrn 

1956; Tarjan and Aragon 1982). The 

pinewood nematode, however, is a facultative parasite capable of living and 

reproducing on both fungi and higher 

plants (mainly Pinus spp. ) (Mamiya 1981, 

1984). Adult pine sawyers (Monochamus 

spp.), which are wood borers, are vectors of the pinewood nematode and other 

species of Bursaphelenchus. The borers 

colonize pine logs held in storage or 

pines weakened or killed by natural or 

manmade stresses (Kellrigl 1971; Webb 

1.909). The pinewood nematode is transmitted by mature beetles feeding on 

shoots of stressed trees or ovipositing 
in dying trees or cut timber (Luzzi and 

others 1984; Wingfield 1983). 

In 1984, the pinewood nematode was 

found infesting wood chips exported from 

North America. Finland placed a permanent embargo on raw softwood shipments 

from the United States, Canada, Japan, 

and other infested regions of the world. 

The other Nordic countries have followed 

suit. The curtailment of southern pine 

chip exports to Nordic countries would 

represent about a $20 million per year 

loss to southern forestry. 

At the shipping terminal (Woodchip 

Export Co.) in Savannah, GA, chips of 

southern pine are accumulated in two 

piles; each contains 15,000 to 20,000 t 

when loading of an ocean vessel begins 

(fig. 1 Normally, it takes 6 to 8 

weeks to accumulate the required tonnage 

of wood chips (about 35,000 t). An 

estimated 70 percent of the chips are 

from slash pine (Pinus elliottii Engelm. 

var. elliottii) and 30 percent are from 

loblolly pine (P. taeda L.) (Wayne 

Stubbs , ~ersonai communication) . 

The development of micro-organisms 

in the chip piles is mainly governed by 

temperature, The temperature in a chip 

pile depends on the ambient temperature, 

the size and compaction of the pile, and 

the fines and bark content of the chips 

(Bergman 1985). Regardless of the 

ambient temperature, the interior of the 

0 pile rapidly rises to 60 C. The temperature of the outer layer, or shell of 

the pile, is usually below the ambient 

temperature (Bergman 1985; Rothrock and 

others 1961; Saucier and Miller 1961; 

Schmidt 1969; Springer and Haljny 1970). 

The initial spontaneous heating in piled 

wood chips is attributed primarily to 

heat released by respiration in living 

sapwood cells (Bergman 1985; Springer 

and Haljny 1970). 

Figure 1.--Southern pine chips (38,000 t) ready for shipment to Sweden. 
To develop strategies for controlling the pinewood nematode in southern 

pine chips, information is needed on the 

biology of the organism in this unique 

ecological niche. The studies described 

here were undertaken to determine: 

What types of chips harbor the 

nematode. 

* Effects of temperature on the reproduction of the nematode in chips. 

* Influence of bluestain fungi in chips 

on nematode populations. 

Influence of chip moisture on nematode 

population dynamics. 

Effects of anaerobic conditions on 

nematode survival. 

Effects of covering chip piles with 

plastic on the thermodynamics of the 

pile, as well as changes in nematode 

populations. 

e Pathogenicity to pine seedlings. 

Methods and Materials 

Nematode Assay 

Nematodes were extracted from chips 

by the pie-tin technique of Kable and 

Mai (1968). Samples were incubated for 

18 to 20 hours at 25 OC and collected 

on a 325-mesh screen. The extracted 

nematodes were concentrated in 10 mL of 

deionized water. Nematodes in a 1-mL 

aliquot were counted under a Wild dissecting microscope and expressed as the 

number per gram of fresh wood weight. 

In some experiments, the population 

densities were increased in chip lots by 

spraying the chips with a pinewood nematode suspension. The population of the 

pinewood nematode used in these experiments was originally isolated from 

Virginia pines (Pinus virginiana Mill.) 

in a seed orchard in Alabama. It was 

reared on Botrytis cinerea Pers, ex Fr. 

growing on potato-dextrose agar at 25 

C. Nematodes were extracted from 7- 

to 10-day-old cultures by the pie-tin 

technique, then collected on a 325-mesh 

screen. 

Chip types. Samples of the following chip types were screened for nematodes: (1) Fresh--Chips collected from 

truck or railcar loads prior to loading 

on the pile. A large percentage of the 

chips were discolored by the bluestain 

fungus. (2) Shell--Chips, also discolored, were collected from the outer 

layer of the pile. (3) Interior--Chips 

were collected from the interior of the 

0 pile where the temperature was 60 C. 

(4) Foundation--Chips that were darkstained, had accumulated on the ground 

over several years, and had served as 

the base for the new chip pile. 

Ten 100-g samples of each of the 

chip types were placed in polyethylene 

interlock seal bags (zip-lock) and 

0 incubated at 25 C for 2 and 8 weeks 

(5 samples per incubation period). In 

addition, ten '75-g samples of each of 

interior, fresh, and foundation chips 

were mixed with 25 g of shell chips, and 

incubated at 25 OC for 2 and 8 weeks. 

At the end of the incubation periods, 

the samples were assayed for nematodes. 

Data were subjected to split-plot 

analysis of variance, with time as the 

main plot. 

Temperature. The population density 

of the nematodes in 6 kg of fresh southern pine chips was increased by spraying 

the chips with 200 mL of a suspension 

containing 1,250 pinewood nematodes/mL. 

These infested chips were mixed and 

stored in a plastic bag at 25 OC for 1 

week, These chips were then divided 

into 50-g samples, placed in zip-lock 

bags, and incubated at 25, 30, 40, 50, 

and60°c. After1, 2, 3, 4, and5 

days, two samples (replications) were 

removed from each incubator and assayed 

for pinewood nematodes, Data were subjected to split-plot analysis of variance procedures, with temperature as the 

main plot. 

In a second temperature study, 6 kg 

of wood chips were heated in an oven at 

60 OC for 24 hours, spread on a table, 

and sprayed with 200 mL of a suspension 

containing 1,000 pinewood nematodeslml. 
These infested chips were incubated at 

25 OC for 2 weeks and then divided 

into 50-g samples placed in zip-lock 

bags. The samples ~ere~incubated at 25, 

30, 35, 40, 44, and 48 C. After 

incubation periods of 1, 3, '7, 14, and 

21 days, four samples at each temperature were assayed for the pinewood 

nematode. Data were subjected to splitplot analysis of variance, with temperature as the main plot. 

Wood chips naturally infested with 

the pinewood nematode were incubated for 

4 days at 25 and 40 OC. Each treatment was replicated three times, and the 

sample size was 75 g. Aliquots of the 

collected nematodes were examined with a 

compound microscope, and the proportions 

of males, females, and juveniles were 

determined for l5O nematodes at each 

incubation temperature. 

Fifty-g samples of 10 lots of chips 

were taken from truck and railcar shipments to the terminal during July 1-5, 

1985. They were incubated in zip-lock 

bags at 25 and 40 OC for 4 days. The 

samples were assayed for the pinewood 

nematode at the end of the incubation 

period, and the data were compared by 

- t-test for paired samples. 

To study effects of high temperatures on the pinewood nematode, 50-g 

samples of naturally infested chips were 

placed in zip-lock bags and incubated at 

50 and 60 OC for 1, 2, 3, 5, 1, and 

24 hours. Each treatment combination 

was replicated six times. The nematode 

population density was determined at the 

beginning of the experiment by assaying 

three 50-g samples. The treatment 

samples were assayed for the pinewood 

nematode at the end of each incubation 

period. The data were not subjected to 

statistical analysis. 

To determine the effects of low 

temperature on nematode density, 3.6 kg 

of chips--returned to the United States 

following a shipment to Sweden--were 

divided into 50-g samples. Samples were 

placed in zip-lock bags an8 incubated at 

-20, 0, 5, 10, 15, and 20 C. After 

incubation periods of 1, 3, 7, or 14 

days, three samples from each temperature were assayed for the pinewood 

nematode. Data were subjected to splitplot analysis of variance, with time as 

the main plot. 

An experiment was also conducted to 

determine the effect of temperature on 

the pinewood nematode in autoclaved 

southern pine chips. Approximately 2.4 

kg of chips were autoclaved for 45 

minutes, allowed to cool, then dipped in 

a suspension of conidia that had been 

collected from cultures the bluestain 

fungus, Cergtocystis sp., grows on malt 

agar at 25 u~. The conidia were 

washed from three culture plates and 

suspended in 2 L of sterile deionized 

water. After dipping, the chips were 

drained and incubgted in the laboratory 

for 3 days at 25 C. They were spread 

on a plastic tray and evenly sprayed 

with a 100 mL suspension of pinewood 

nematodes (1,00O/mL). Four days later, 

these infested chips were divided into 

50-g samples, placed in zip-lock bags 

and incubated at 25, 30, 35, and 40 06 

for 1, 3, 6, and 9 days. There were 

three replications of each treatment 

combination. Data were subjected to 

two-way analysis of variance. 

Bluestain. On July 1-5, 1985, a 

total of 38 truck and railcar shipments 

to the exporter were randomly sampled. 

Individual chips from 100-g subsamples 

were separated according to presence or 

absence of bluestain. The resulting 

subsamples were reweighed, and the percentage of weight by chips with or without bluestain was determined for each of 

the 38 lots. The samples were then 

readjusted so that each group had a 

fresh weight of 50 g. These adjusted 

samples were assayed for nematodes, and 

data were compared by the t-test for 

paired observations. 

Ten of the 38 lots that had been 

divided into subsamples containing chips 

with or without bluestain were incubated 

in zip-lock bags at 40 OC for 6 days 

and then assayed for nematodes. The 

sample weight was 50 g, and each treatment combination was replicated twice. 

Data were subjected to two-way analysis 

of variance and &-test for paired 

observations. 
Chip moisture. The influence of 

chip moisture on nematode population 

density was investigated. From a 1.2-kg 

sample of chips naturally infested by 

the pinewood nematode, 25-g samples were 

randomly selected and placed in tared 

weighing dishes. The resulting 45 samples were placed in desiccators containing calcium chloride, water, or neither 

(150samples/treatment) and incubated at 

35 C. Six samples were assayed for 

nematodes to determine the base level. 

After incubation periods of 2, 5, 9, 13, 

and 21 days, three samples were removed 

from each container, weighed to determine the percentage of moisture loss, 

and assayed for nematodes. Data were 

subjected to two-way analysis of variance and regression. 

Anaerobic environment. Effects of 

an anaerobic environment (C02) on 

nematode population densities in pine 

chips were studied in GasPak Plus 

Anaerobic Jar System (BBL Microbiology 

Systems, Cockeyville, MO 21030). In one 

experiment, 39-g samples of six randomly 

selected lots of chips were placed in. 

petri dishes, sealed in a GasPak, and 

incubated at 38 OC for 3 days. The 

control samples were concurrently incubated in an aerobic environment at the 

same temperature. At the end of the 

experiment, the samples were assayed for 

nematodes, and the data were compared by 

- t-test for paired observations. 

In a second experiment, the base 

nematode population densities were 

determined. Then 39-g samples of 10 

randomly selected lots of chips were 

incubated for 5 days at 38 OC in an 

aerobic environment and transferred to 

an anaerobic environment for 3 days. 

Two sets of controls, incubated in an 

aerobic environment at 38 OC for 5 to 

8 days, were included. The samples were 

assayed for nematodes at 5 or 8 days. 

Data were compared by t-test for paired 

observations. 

Chip piles. A study was conducted 

to learn about thermodynamics of piled 

chips and the effects of heat on nematode population dynamics in chip piles. 

Two cone-shaped chip piles, 6 m in diameter at the base and 2.4-m height were 

constructed. Chips were taken from the 

shell of a nearby main pile in Savannah 

with a front loader. The piles were 

constructed with four layers (fig. 2). 

Each layer was implanted with 450 g chip 

samples infested with nematodes and 

enclosed in plastic hardware cloth. The 

numbers of samples for layer 1 (bottom), 

2, 3, and 4 (peak) were 4, 3, 2, and 1, 

respectively. 

COVERED 

UNCOVERED 

Figure 2.--Diagram of simulated chip piles showing 

the location of the pinewood nematode-Infested 

chip samples (A-J) and the temperature probes (0). 

Five thermistor probes were placed 

at different locations in the piles and 

connected to an analog telethermometer 

(Model 47, Yellow Springs Instruments, 

Inc,, Yellow Springs, OH) housed in a 

standard weather station enclosure 

located between the piles. An eleventh 

probe measured ambient temperature. One 

of the piles was covered with a sheet of 

4-mil plastic; the other was left uncovered. The temperatures were recorded 

Monday through Friday at 0800, 1200, and 

1600 hours, 

The study was established August 26, 

1985, and terminated September 12, 1985. 

At the conclusion of the study, the chip 
implants were removed from the piles and 

assayed for nematodes by screening three 

50-g subsamples from each bag. Data 

were compared for covered and uncovered 

piles and location within the pile by 

- t-test for paired observations. 

Pathogenicity. The pinewood nematodes extracted from 10 lots of chips 

that hgd been incubated for 4 days at 25 

or 40 C were used in a preliminary 

study on pathogenicity to 2-year-old 

seedlings of slash pine in a greenhouse. 

Seedlings were grown in plastic flats 

(33x13~11 cm) containing a mixture of 

soil, pine bark, and sand (2:1:1, v/v). 

Each flat contained 10 seedlings that 

had been transplanted when they were 1 

year old. 

The 20 populations used as inocula 

were adjusted to 1,000 nematodes/mL. 

Since the inocula were from naturally 

infested chips, other nematode species 

and associated organisms were not 

removed from the inoculum suspension. 

To inoculate each seedling, an area 

(3 mm x 10 mm) of bark on the main stem 

was removed, then a piece of sterile, 

absorbent cotton containing 200 pinewood 

nematodes was attached with Parafilm 

over each stem .wound, Five seedlings 

were inoculated with each nematode 

population. Equal numbers of wounded 

but uninoculated seedlings served as 

controls. The study was initiated in 

late July 1985 and concluded 16 weeks 

later. 

At the conclusion of the experiment, 

seedling mortality was recorded. Then 

all seedlings were sampled for nematodes 

by extracting from 25-mm sections of the 

stem directly below the inoculation 

point and at the base of the seedling. 

Sections were incubated in 5 mL of water 

in separate test tubes at 25 OC for 20 

hours. Extracted nematodes were not 

counted. Mortality data (25 vs. 40 OC) 

were compared by using the t-test for 

paired observations. 

Results 

Chip types. Most of the nematodes 

extracted were saprobic species rather 

than pinewood nematodes. An analysis of 

variance of the total nematode or pinewood nematode population densities 

showed that the experimental variation 

was caused by chip types or mixes and 

not by incubation period or treatment 

interactions. There was, however, a 

notable increase in general nematode 

densities in fresh chips between week 2 

and week 8 of incubation (table 1). Of 

the four main chip types studied, nematodes were most numerous in shell and 

fresh chips. There were significantly 

(P=0.01) fewer nematodes in the interior 

and foundation chips, The highest total 

nematode densities were found in the 

treatments having shell chips mixed with 

interior or fresh chips prior to incubation. Also, the nematode levels in 

mixtures exceeded those that would have 

been predicted based on the levels 

recorded for the individual chip types. 

The predicted mean nematode count for 

interior + shell chips was 88 nematodes/g fresh wood weight (C0.5 x 751 + 

[348 x 25])/100, and for fresh + shell 

chips it was 351 nematodes/g ([75 x 751 + [348 x 75])/100, whereas the actual 

count was 789 and 743 nematodes/g, 

respectively, Mixing shell chips with 

foundation chips apparently did not 

influence nematode population densities 

in the same manner, because the actual 

count of 66 nematodes/g was less than 

the predicted count of 120 nematodeslg 

(C57 x 751 + 1348 x 251)/1004 

The analysis of variance showed that 

population densities of the pinewood 

nematode in the chips were related to an 

interaction of chip types with incubation period. This interaction was 

reflected primarily in the significant 

(P=0.01) increase in pinewood nematode 

counts in the fresh chips between the 2- 

and 8-week incubation periods and the 

decrease in counts in the interior + 

shell mix during this period, The 

counts in other treatments (chip types 

or mixes) were not affected by incubation period. Pinewood nematodes were 

recovered from only shell and fresh 

chips and were not found in interior or 

foundation chips. 

Temperature. Temperature had a 

marked influence on the reproduction and 
Table 1.--Assay of wood-chip types and mixes for pinewood ncmatode and other nematodes 

after incubation at 25 OC for 2 and 8 weeks 

All ncmalodes 

after incubation for-- 

Pinewood nematodes 

aftcr incubation for-- 

Types of wood a b 

chips and mixes 2 wk 8 wk Mean 2 wk 8 wk Mean 

Shell (S) 130 565 348 13 14 CD 12 CD 13 

Interior (I) 0-7 0.3 0.5 C 0 D 0 D 0 

Presh (PR) 3 0 676 353 l.3 5 CD 32 B 18 

Foundation (PUN) 4 3 7 1 57 nc 0 D 0 D 0 

3.+s 789 608 698 A 55 A 9 CD 32 

PR + S 7'1 3 l.O3/i 888 A 10 CD 18 ilC 1 11 

PDN + S 5 1 7 1 66 JIC 3 CD 8 CD 6 

a 

Chip types: Fresh--chips collccted from truck or railcar loads prior to loading on the 

pile. Shell--chips collccted from the outer layer of the pile. Interior--chips collected 0 

from the interior of the pilc where the temperature was 60 C. Foundation--old chips 

that served as the base for the new chip pilc. For S. I. FH, and PUN, the sample size was 

100 g. The mixes consisted of 75 g of I. Ffi. or FDN chips plus 25-g S chips. There were 

five replications of each treatment combination. 

b 

Within columns, means followed by the same fetter do not differ significantly (P=0.01) 

according to Duncan's multiple range test. 

survival of the pinewood nematode in 

these studies. In the initial experiment, the nematode population density at 

40 OC increased significantly (P=O. 01 ) 

over time (fig. 3). Afte; 5 days, the 

population density at 40 C was 15 

times the average density for 25 and 30 

0 C. After 1 day, the number of nematodes in chips incubated at 50 and 60 

0 C was zero. At 25 and 30 OC, there 

was no significant change in the population densities during the period of the 

experiment. In naturally infested wood 

chips, 102 nematodes/g were extracted 

from samples incubated for 4 days at 25 

0 C and 646 nematodes/g at 40 OC. At 

25 OC, 13, 17, and 70 percent of 150 

nematodes examined were males, females, 

and juveniles, respectively. At 40 OC, 

10, 29, and 61 percent were males, 

females, and juveniles, respectively. 

Only the propagative forms of the nematode were observed, 

In the second experiment, the analysis of variance showed that experi~nental 

variation was influenced by the time x 

temperature interaction (P=0.01). At 25 

and 30 OC, the nematode densities 

declined from 55 nematodeslg at day 1 to 

3 nematodes/g at day 21 (fig. 4). At 35 

C, the population densities were 1.18 

and 122 nematodeslg on days 3 and 7, 

respectively, and then declined to lower 

densities at later periods. At day 21, 

however, the nematode densities were 

still 2.6 times greater than those in the 

25 and 30 OC treatmegts. The nematode 

densities in the 40 C treatment 

increased to 250 nematodeslg by day 3 and 
Figure 3.--Effect of moderate temperatures (25 to 

0 

60 Oc) on pinewood nematode population 

- 50/ 

60°C 1 I 1 I 1 

development in southern pine chips over a 5-day 

I 2 3 4 5 

INCUBATION PERIOD (DAYS) 

incubation period. 

1 3 5 7 9 11 13 I5 17 19 21 

INCUBATION PERIOD (DAYS) 

Figure -%.--Effect of moderate temperatures (25 to 0 

48 C) on pinewood nematode population 

development in southern pine chips over a 21-day 

incubation period. 

then declined to lower densities at later 

periods. After day 21, these densities 

approached those in the 35 OC treatment. The temperature of the incubator 

set at 44 OC crept to 45 OC between 

days 1 and 3. At day 1, the average 0 nematode density in the 44 C treatment 

was 96/g, but it declined to 6.2 nematodes/g by day 3 when the temperature had 

increased to 45 OC, and remained log 

until the end of the study. At 48 C, 

the population density averaged 0.7 nematodes/g over time, The pinewood nematode 

increased significantly over time at 

temperatures between 35 and 44 OC in 

this study, with optimum for nematode 

0 reproduction in the 40 C treatment. 

In the temperature experiment where 

the chips had been autoclaved and inoculated with the bluestain fungus prior to 

being infested with the pinewood nematode, the analysis of variance showed 

experimental variation was due to the 

main effects (time and temperature) . Among all four incubation temperatures, 

the nematode population density declined 

at day 3, then returned to the base level 

by day 6 (table 2). The mean number of 

nematodes extracted over time was 89 

nematodes/g at 35 OC, which was significantly (P=0.01) greater than the 59 

Table 2.--Effect of temperature on population 

development of pinewood nematode in autoclaved 

chips inoculated with Ceratocystis sp. and 

pinewood nematode 

0 

Temperature ( C) 

Incubation 

period a (days) 25 3 0 35 40 Mean 

1 87 75 104 6 1 81 A 

3 3 0 2 1 38 35 31 B 

6 62 79 114 65 80~ 

9 58 72 loo 53 71 A 

a Mean 59 n 62 n 89 r 54 n 

a 

Within temperature and incubation periods, 

means followed by the same letter do not differ 

significantly (P=O.Ol) according to Duncan's 

multiple range test. 
Table 3.--Pinewood nematodes extracted from 10 0 

lots of wood chips incubated at 25 and 40 C for 

4 daysa 

Table &.--Effect of high temperature on pinewood 

nematode populations in wood chips 

0 

Temperature ( C) 

Temperature 

Lot Change 0 

number 25 c 40 OC (40/25 Oc) 

~ematodeslg fresh weight 

a 

For 50-g samples extracted for 18 to 20 hours 0 

at 25 C. 

b 

Means are significantly different (g=0.01) 

according to t-test for paired observations. 

nematodes/g at 25 OC, the 62 nematodes/g at 30 Ec, and the 54 nematodes/g at 40 C. The optimum temperature for reproduction of nematodes in 

this study was 35 OC. 

When samples of 10 lots of naturally 

infested wood chips were incubated at 25 

and 40 OC for 4 days, there was noticeable lot-to-lot variation in response to 

temperature (table 3). In lot 3, for 

example, there was a threefold increase 

in nematode levels between 25 and 40 

0 C. In lot 9, however, the densities 

0 

were higher at 25 C than at 40 OC. 

Nevertheless, 10 lots were significantly 

different (g=0.01), and the average 

increase in the po ulation density 8 between 25 and 40 C was 1.6 times. 

The pinewood nematode tolerated 

temperatures greater than 50 OC for no 

Time 

(hours) 

a 

The base population density at time 0 was 127 

nematodes/g. Each treatment combination was 

replicated three times. Data based on 50-g 

samples of chips extracted for 18 to 20 hours at 0 

20 C. 

b 

Populations expressed as percentage of initial 

(time 0) population density. 

more than a few hours (table 4). After 

1 hour at 50 OC, the nematode population density was 47 percent of the base 

level. After 13 hours, no pinewood 

nematodes were recovered from chip 

samples. After 1 hour at 60 OC, the 

population density was reduced by 99.8 

percent, compared with the base level, 

and after 5 hours, no nematodes were 

recovered from chip samples. 

In a low temperature experiment, the 

analysis of variance showed the experimental variation was attributed to the 

temperature x time interaction (P=O.Ol) 

(fig. 5). Nematodes survived in wood 

chips for only 3 days at -20 OC. The 

population densities were fairly constant over time at 0, 5, and 10 OC. 

At 15 OC, there was an increase in 

nematode densities at days 3 and 7, 

followed by a gradual decrease to day 14. 
1234 5 6 7 891011 121314 

INCUBATl ON PERIOD (DAYS) 

Figure 5.--Effect of low temperatures on pinewood 

nematode population development in southern pine 

chips. 

Bluestain. Pinewood nematodes were 

recovered from 92 percent of the 38 lots 

sampled from July 1-5, 1985. The mean 

percentage by weight of wood chips with 

bluestain was 45 percent. The mean 

number of nematodes/g was 107 for chips 

with bluestain. This number was significantly (P=0.01) greater than the 82 

nematodes/g for chips without bluestain. 

When 10 lots of chips were divided 

into samples with or without bluestain 

and incubated at 40 OC for 6 days, the 

analysis of variance found that experimental variation was due to lots. The 

mean number of 669 nematodes/g extracted 

was somewhat higher than the 624 nematodes/g extracted in a previous study 

(see table 2). Based on a t-test for 

paiped observations, there were significantly (P=0.01) more nematodes in the 

bluestain chips (747 nematodes/g) than 

in chips without bluestain (591 nematodesfg). These po~ulation densities 

are approximately seven times those at 

40 OC in a previous comparison (107 

and 82 nematodes/g, respectively). This 

result indicates that the effect of 

temperature was additive. 

Chip moisture. Wood moisture markedly affected the population densities 

of nematodes in pine chips (fig. 6). 

After 5 days, the nematode densities in 

the chips declined as the percentage of 

moisture in the chips declined, In the 

control, the nematode level in the chips 

declined from 40 nematodeslg to 26 nematodeslg as the percentage of moisture 

loss reached 22. The nematode level 

also decreased in chips incubated over 

water, 

Anaerobic environment. In the first 

study of pinewood nematode in an anaerobic environment, the population density 

was 157 nematodes/g for chhps in an 

aerobic environment at 38 C after 3 

days. Under anaerobic conditions under 

the same temperature after 3 days, the 

population level was eight nematodes/g. 

In the second experiment, the base 

(time 0) mean nematode density was 10 

nematodes/g for the 10 ch6p samples. 

After 5 and 8 days at 38 C in an 

aerobic environment, the population 

level was 43 and 40 nematodes/g, 

respectively. When chip samples were 

incubated for 5 days in an aerobic 

environment and then transferred to 

anaerobic conditions for 3 days, the 

population density dropped to 4 

nematodes/g. 

CJ WATER ---- % 40 CONTROL em.... a. t 

INCUBATION PERIOD (DAYS) 

Figure 6.--curves showing moisture loss in 

southern pine chips (A) and corresponding changes 

in population levels of pinewood nematode (B) at 0 

35 C over time. 
Chip piles. There were significantly (P=0.01) fewer pinewood nematodes 

(8 nematodesig) in the samples incubated 

in the chip pile covered with plastic 

than in samples from the uncovered pile 

(30 nematodes/g). In the covered chip 

pile, the highest nematode density was 

found in the samplas placed in the shell 

of the lower or first level. The temperature at this location averaged 41 

0 C and never exceeded 45 OC. For 

the rest of the pile, the temperature 

averaged 43.9 OC and exceeded 45 OC 

for 60 to 366 hours, depending on the 

location of the probe. In the uncovered 

pile, the highest nematode density (52 

nematodeslg) was in the center sample of 

the second layer. The temperature of 

the uncovered pile averaged 30.2 OC 

for the five probes. The mean ambient 

0 temperature was 30.2 C. The temperature increase in the covered pile was 

largely due to solar radiation. 

Pathogenicity. In the pathogenicity 

study, 20 percent of the slash pine 

seedlings inoculated with 20 (10 chip 

lots x 2 temperatures) populations of 

the pinewood nematode died within 16 

weeks. There was no significant difference, according to t-test for paired 

observations, between the nematode 

gopulations from chips incubated at 25 

C (22 percent mortality) and 40 OC 

(18 percent mortality). There was no 

lot-to-lot variation. None of the control seedlings died. Pinewood nematodes 

were the only nematodes recovered from 

symptomatic seedlings. 

Discussion 

Nematodes, particularly Rhabditis 

spp. and other saprobic nematodes, are 

commonly found in wood-chip piles. The 

pinewood nematode, a facultative parasite, was recovered from 92 percent of 

the chip samples collected from shipments from suppliers to the Savannah 

terminal in July. Since the living 

cells in fresh sapwood chips retain 

their viability only about 2 weeks at 21 

0 C (Feist and others 1971), the pinewood nematode is largely dependent on 

fungi in chips for nutrition and lives 

saprophytically in these chips for 

longer periods. 

The complex thermodynamics of piled 

chips governs the population dynamics of 

the pinewood and other nematodes associated with southern pine chips. The 

spontaneous heating of the interior 

chips caused by oxidation is sufficient 

to kill all nematodes. The pinewood 

nematode is found in fresh chips and 

other chips that have not been subjected 

to this "self-pasturizing." The population densities of saprobic nematodes 

increased rapidly to high densities when 

interior chips were mixed with shell 

chips. This increase was probably 

caused by the reduction of predators and 

nematophagous fungi in the interior 

chips. Three nematophagous fungal 

species, including Arthobotrys 

oligospora Fres., have been cultured 

from the sapwood of slash pine (Esser 

and others 1983). Because of the spontaneous heating in piled chips, the 

pinewood nematode densities in chip 

piles should normally decrease to low 

levels over time. 

In the present study, the optimum 

temperature range for the reproduction 

of the pinewood nematode in southern 

pine chips was 35 to 40 OC. These 

values are considerably higher than 

those in other reports. Most studies on 

the effects of temperature on the 

population dynamics of the pinewood 

nematode have been done on Botrytis 

cinerea in culture. For example, the 

optimum temperature for the pinewood 

nematode on 51. cinerea has been reported 0 

as 25 C (Dozono and Yoshida 1974). 

Mamiya (1983, 1984) reports that the 

pinewood nematode will not reproduce at 

over 33 OC. Temperature studies using 

mesophilic fungi as a host for the pinewood nematode failed to consider the 

effect of temperature on the growth and 

physiology of the fungus. In chips, for 

example, over 200 species of fungi have 

been isolated, including such common 

thermotolerant fungi as Phaneroehaete 

chrysosporium Burdsall & Eslyn and 

Aspergillus A fumi atus Fres., which grow 

fastest at 35 to 40 C (Bergman 1985; 

Burdsall and Eslyn 1974; Hulme 1979). 

These thermotolerant fungi may be a 

suitable food source for the pinewood 

nematode and explain why the nematode 

reproduces well at this temperature 

range in wood chips,. 
Although the o timum temperature 8 

range of 35 to 40 C was noted in 

several experiments, one study indicated 

that the pinewood nematode populations 

in different lots of southern pine chips 

did not always respond in tge same 

manner to incubation at 40 C. In 

some lots, the chips were probably less 

than 2 weeks old and the pinewood nematodes were probably feeding on the 

epithelial cells of the resin ducts 

(phytophagous). In other lots where the 

nematodes were mycophagous, reproduction 

was likely influenced by the presence or 

absence of thermotolerant fungi. 

At temperatures above 45 OC, the 

population densities of the nematode in 

pine chips rapidly declines. To illustrate this temperature x time relationship, it takes about 13 hours for thg 

population to decline to zero at 50 C 

but only 1 hour at 60 OC. Unfortunately, it may not be economically 

feasible to use such a high temperature 

to control the pinewood nematode in wood 

chips because of the limited heat conductivity of wood and the rate the chips 

are loaded on ships (600 to 700 t/h). 

One estimate suggests it would take over 

33 million Btuts/hoto raise 10 t of wet 

wood chips/min 20 C (W.E. Buske, 

Wolverine Corp,; personal communication). The energy costs and high 

associated capital investment have ruled 

this approach out for now. 

The presence of bluestain in chips 

is an indicator of infestation of pines 

by bark beetles, but it cannot be used 

accurately to quantify nematode infestation of shipments from suppliers. 

Because of the frequent association of 

sawyers with southern pine (Dendroctonus 

frontalis Zimm.) and engraver (Ips 

calligraphus (Germ.) beetles (Coulson 

and others 1976; Miller 1984), shipments 

with bluestained chips frequently contained pinewood nematodes. Quite often, 

however, lots with a high percentage of 

chips with bluestain failed to yield 

nematodes. Pinewood nematodes have also 

been recovered from samples in which 

there are few chips with bluestain, if 

any. 

Fresh chips with bluestain yielded 

significantly more nematodes than those 

without bluestain. The magnitude of the 

difference (1.3 times), however, was not 

as great as might have been expected for 

this mycophagous organism. Other fungi 

that colonize chips or wood of trees 

attacked by sawyers and bark beetles 

(Bergman 1985; Burdsall and Eslyn 1974; 

Luzzi and others 1984; McCrawley and 

others 1980) may prove to be more 

important in the ecology of pinewood 

nematodes than Ceratocystis spp. 

Gliocladium virens Miller (McCrawley and 

others 1985). for example, has been 

isolated from nematode-infested slash 

pines and is an excellent host for pinewood nematodes. Because of the thermogenic environment of piled wood chips, 

the role of thermotolerant fungi, such 

as Phanerochaete chrysosporium and 

Aspergillus fumigatus, in the development of pinewood nematodes needs to be 

investigated. 

Pinewood nematodes are intolerant of 

anaerobic conditions at 38 OC. Since 

they are aerobic or semi-aerobic invertebrates, fumigants such as phosphine 

may be effective in controlling nematodes in wood chips stored in the holds 

of ocean vessels. Preliminary tests 

with phosphine show promise for controlling the nematode in wood chips (J.G. 

Leesch, ARS Stored Product Insects 

Research and Development Laboratory, 

Savannah, GA.; personal communication). 

It is doubtful whether southern pine 

chips could serve as a source of inoculum for the establishment of pine wilt 

disease in countries that import them. 

Unless the nematode is transmitted by a 

vector, it is destined to be digested in 

the pulping process. The basic biological requirements of pine sawyers cannot 

be met by wood chips (Webb 1909; 

Hellrigl 1971). 

The author thanks Bergitta Kemp and 

Wayne Stubbs of Savannah Sales, Inc., 

and the staff at Woodchip Export Company 

for assistance in this study. 

The study was partially funded by a 

grant from the Georgia Forestry 

Commission. 
Literature Cited Kable. P.F.; Mai, W.P. Influcncc of soil moisture 

on Pratylenchus penetrans. Ncmatologica 

Ih:IOI.-I22; 1968. 

Uergman, asten. Deterioration and protection of 

pulpwood chips in outdoor storage (Ncdbrytning 

och skydd av massavedsflis vid lagring utomhus). 

Rep. 170. Uppsala, Sweden: Swedish University 

of Agricultural Science, Department of Forest 

Products; 1985. 95 pp. 

Burdsall, H.II., Jr.; Eslyn, W.E. A new 

Phanerochacte with a Chrysosporium imperfect 

stage. Mycotaxon 1:123-133; 1974. 

Coulson, R.N.; Mayyasi, A.M.; Poltz, J.L.; Main, 

F.P. Interspecific competition between 

Monochamus titillator and Dendroctonus 

frontalis. Environmental Entomology 5:235-247; 

1976 

Dozono, Yasou; Yoshida, Nariaki. Application of 

logistic curve for the population growth of pine 

wood nematode, Uursaphclenchus lignicolus, on 

the cultures of Botrytis cincrea. Journal of 

the Japanese Forestry Society 56:146-148; 1974. 

Dwinell. L. David; Barrows-Broaddus, Jane. Pine 

wilt and pitch canker of Virginia pine in seed 

orchards. In: Proceedings of the 17th Southern 

Forest Tree Improvement Conference; 1983 June 

7-9; Athens, GA. Southern Forest Tree Improvement Committee sponsored publication 39; 

1983:55-62. 

Esser, R.P.; Wilkinson. R.C., Jr.; IIarkcom. K.J. 

Pinewood nematode (Bursaphelenchus xylophilus) 

survey in Plorida. Proceedings of the Soil and 

Crop Science Society of Plorida. 42:127-132; 

1983 - 

Peist, W.C.; Springer. E.L.; Ilajny, C.J. 

Viability of parenchyma cells in storcd green 

wood. Tappi 54:1295-1297; 1971. 

1Iellrigl. K.G. Die Uionomie der curopaischen 

Monochamus-Arten (Coleopt.. Cerambycid) und ihre 

Bedeutung fiir die Forst- und Ifolzwirtschaft. 

Bolzano, Italy: Centro di Entomologia Alpina e 

Porestale del Consiglio Nazionale dclle 

Richerche; Publlicazionc N. 154; 1971:367-509. 

Iiulme. M.A. Outside chip storagc: dctcrioration 

during outside chip storage. In: Ifatton, J.V., 

ed. Chip Quality Monograph. Pulp and Paper 

Technol. Ser. 5; 1979:213-244. 

Luzzi, M.A.; Wilkinson, R.C.. Jr.; Tarjan, A.C. 

Transmission of the pincwood nematodc, 

Uursaphelenchus xylophilus, to slash pinc trees 

and log bolts by a cerambycid bcetle, Monochamus 

titillator, in Plorida. Journal of Ncmatology 

16:37-40; 1984. 

Mamiya, Y. Pathology of the pine wilt disease 

caused by ~ursaphclcnchus xylophilus. Annual 

Keview of Phytopathology 21:201-220; 1983. 

Mamiya, Y. Thc pine wood nematodc. In: Nicklc, 

W.R., ed. 1984. Plant and insect nematodes. 

New York: Marcel Dckker, Inc.; 1984:589-625. 

Masscy. Calvin L. Biology and taxonomy of ncmatodc parasites and nssociatcs of bark beetles in 

the United States. Agric. IIandb. h46. 

Washington. DC: U.S. Departmcnt of Agriculture. 

Porcst Service; 1974. 233 pp. 

McCrawley. E.C.; Jones, J.P.; Birchfield, W. 

Reproduction of I$ursaphclcnchus lignicolus on 

fungi isolated from 2. lignicolus-infested pine 

trees. Journal of Ncrnatology 12:231; 1980. 

Abstract. 

McCrawley, E.C.; Winchcll. K.L.; Jones. J.1'. 

Land others]. Population dcvclopmcnt and influence of ~ursaphclcnchus xylophilus on 

Gliocladium vircns. Journal of Ncmatology 

17:69-76; 1985. 

ilc M Effect of exclusion of inscct 

associates on Ips calligraphus (Ccrm.) 

(Colcoptera: Scolytidac) broad emergence. 

Zeitschrift fur AngcwandLc Entomologic 

97: 298-3014 ; 1984. 

Rothrock. C.W., Jr.; Smith. W.H.; Lingrcn, R.M. 

The effects of outside storage on slash pine 

chips in the South. Tappi 44:65-73; 1961. 

Huhm, W. Die Nematodcn dcr Ipidcn. 

Parasitologishc Schriftcnreische 6; 1956. 

437 PPSaucier. J.R.; Millcr. R.L. Deterioration of 

southern pinc chips during summer and winter 

storage, Porcst Products Journal 11:371-379; 

1961. 
Schmidt, P.L. Observations on spontaneous heating 

toward combustion of commercial chip piles. 

Tappi 52:1700-1701; 1969. 

Springer, E.L.; Haljny, G.3. Spontaneous heating 

in piled wood chips. I. Initial mechanism. 

Tappi 53:85-86; 1970- 

Tarjan, A.C.; ~ragdn, Carlos Baeza. An analysis 

of the genus Bursaphelenchus Puchs, 1937. 

Nematropica 12:121-144; 1982. 

Wcbb, J.L. The southern pine sawyer. Bull. 58. 

Washington, DC: U.S. Department of Agriculture, 

Bureau of Entomology; 1909:41-56. 

Wingfield. M.J. Transmission of pinewood nematode 

to cut timber and girdled trees. Plant Disease 

67(1):35-37; 1983- 

Wingfield, M.J.; Blanchette, R.A.; Nicholls, T.W.; 

Hobbins, K. The pine wood nematode: a compariso11 of the situation in the United States and 

Japan. Canadian Journal of Forest Research 

12:71-714; 1982. 
Dwinell, L. David 

Ecology of the pinewood nematode in southern pine chip piles. 

Res. Pap. SE-258. Asheville. NC: U.S. Department of 

Agriculture, Porest Service, Southeastern Porest Experiment 

Station; 1986. 14 pp. 

The optimum temperature range for pinewood nematodes in southern 

pine chips was 35 to 40 OC. Nematode populations declined at 0 0 

temperatures of -20 C, at temperatures above 45 C, and in 

anaerobic environments. Wood moisture content and presence of 

bluestain fungus also influenced nematode populations. 

KEYWORDS: Bursaphelcnchus xylophilus, temperature, bluestain, pine 

wilt, slash pine, loblolly pine, pathogen. 

Dwinell, L. David 

Ecology of the pinewood nematode in southern pine chip piles. 

Res. Pap. SE-258. Asheville. NC: U.S. Department of 

Agriculture, Forcst Service, Southeastern Forest Experiment 

Station; 1986. 14 pp. 

The optimum temperature range for pinewood nematodes in southern 

pine chips was 35 to 40 OC. Nematode poptflations declined at 0 

temperatures of -20 C. at temperatures above 45 OC. and in 

anaerobic environments. Wood moisture content and presence of 

bluestain fungus also influenced nematode populations. 

KEYWORDS: Bursaphelenchus xylophilus, temperature, bluestain, pine 

wilt. slash pine, loblolly pine, pathogen.