Reprinted from PHYTOPATHOLOGY, Vol. 58, :\'0. 2, 262-26."
Printed in U. S. A.
An Undescrihed Gall Midge Leaf Spot
of Balsam Poplar
H. S. Whitney and J. A. Baranyay
Canada Department of Forestry and Rural Develop­
ment, Forest Research Laboratory, Calgary, Alberta.
We wish to thank H. A. Tripp, who identified the
larva as that of a gall midge (Cecidomyiidae).
A hitherto undescribed leaf spot of balsam poplar
(PoPUlus balsamifera L.) associated with a gall midge
(Cecidomyiidae) was first observed in Waterton Lakes
National Park, Alberta, in 1963. Subsequent collections
indicated its wide distribution in Alberta. Only a minor
portion of the foliage of a tree was affected and there
was no indication of premature leaf fall. Observations
and culture studies were undertaken because, although
this spot resembled those caused by pathogenic fungi,
it did not fit an existing description.
Selected saplings of balsam poplar were tagged in
mid-June 1965 and 1966 in the Kananaskis Forest Ex­
perimental Station, Alberta. Approximately 100 selected
leaves with symptoms were observed frequently each
summer. Attempts were made to obtain pure cultures
of fungi growing in the leaf spot tissue from Kananaskis
and from elsewhere in the Alberta-Territories region.
February 1968
in the epidermis and where a larval skin was often
present. Although most spots were localized and re­
mained unchanged in size until leaf drop, some spots
darkened occasionally, developed a very irregular
margin, and became larger (Fig. 7). Typically, however,
necrotic spots were circular, dry, dark brown, sur­
rounded by a faint chlorotic halo, and often had fungus
spores in the area of the papilla. The average diameter
of 34 localized necrotic spots from 25 randomly selected
leaves was 5.5 mm.
Many different fungi were frequently cultured from,
or observed on, the necrotic spots. The numbers of
microorganisms isolated from 110 necrotic spots were:
Cladosporium, 40; Alternaria, 9; Botrytis, 4; Candida,
4; Phama, 3; Penicillium, 2; Septaria, 2; Fusidium, 2;
Trichoderma, 1; Fusarium, 1; unidentified fungi, 21;
yeast, 1; and bacteria, 4. Sometimes two or three fungi
were found in the same spot. However, 25 necrotic spots
did not produce microorganisms at all. Also, eight
chlorotic spots, and larvae from seven additional spots,
were free from microorganisms except one larva that
produced a species of Candida. Leaf pieces containing
infected localized spots produced fungus growth only
from the necrotic tissue. By contrast, leaf pieces with
enlarged, blackened necrotic patches produced fungus
growth from green or chlorotic tissue 1-2 mm beyond
the margin of necrosis.
Adaxial epidermal and palisade cells and their nuclei
Leaf pieces containing affected and unaffected tissue
were excised aseptically, washed for 30 min in running
tap water, rinsed for 5 min in 10% sodium hypochlorite,
patted dry, and placed on 2% water agar in petri plates.
and nucleoli were noticeably enlarged near the papilla.
There was no evidence of cell division, and mycelium
was not seen.
Fungi were identified from pure cultures on 2% potato­
dextrose agar and from fructifications on leaf tissue.
Cecidomyiid larvae were treated in the same way as
The effect of injury such as might be caused by larvae
feeding on the leaf was investigated. Six young, clean
leaves were injured by making 10 pin punctures in an
leaf pieces.
Fresh material was fixed in formalin:acetic acid: 70%
ethanol (95:5:5) for cytological study of spot develop­
area 2 mm in diam. A drop of sterile water was placed
on such an injured surface on each of two leaves on
ment. The fixative was replaced with water and the
tissue embedded in 1% gelatin in a cryostat at -20 C.
Sections 10!l thick were viewed microscopically with
phase contrast illumination.
bag. The bags were removed 4 days later and the leaves
examined after an additional 10 days. On all leaves, the
The first conspicuous symptom of the leaf spot was
observed near the end of June as a tiny chlorotic
papilla, approximately 1.5 mm in diam and 1.0 mm
high (Fig. I-A), which protruded adaxially with an
abaxial concavity. The concavity was partially filled
with a clear sticky fluid within which lay a cecidomyiid
larva (Fig. 2). Papillae occurred singly or in groups.
Three to 4 days later each papilla or group of papillae
was surrounded by a chlorotic zone with a brown to
black perimeter (Fig. I-B). On dried specimens the
papilla was less chlorotic than the surrounding tissue
three trees, and the leaves were enclosed in a plastic
margin of each puncture was necrotic but there was
neither chlorosis nor papillae produced, nor apparent
fungal infection of the surrounding tissue.
We are postulating that the cecidomyiid larva in­
jured the leaf tissue and thereby rendered it liable to
colonization by fungi. The consistent association of a
cecidomyiid larva, from the earliest stages of spot
development until death of the tissues in the spot, and
the lack of any one culturable microorganism intimately
associated with this leaf spot indicate that the larva
was the primary cause. This view is strengthened by
the frequent absence of fungi from larvae, from leaf
seen in both fresh and dried developing spots (Fig. 4).
tissue in early stages of spot development, and from
older necrotic spots. The nature of the injury to the
leaf tissue is unknown, but clearly it is not due to
fungus infection nor is it likely due entirely to feeding
Segments of necrotic tissue then began to appear within
the spot, and by approximately 2 weeks after the first
symptoms the spot was completely necrotic (Fig. 5).
of larvae. It may be caused by a larval toxin, a phyto­
alexin, or a virus. Although fungus colonization of in­
fected spots was usually limited to the spot, it some­
within the spot, thus resulting in a green island (Fig. 3).
With transmitted light, a marked vein clearing was
During the later stages of necrosis the cecidomyiid
times extended into surrounding tissue. Growth of fungi
larva left the leaf and the papilla flattened. The margin
of necrotic spots was irregular, especially abaxially
beyond the spot and throughout the leaf would be an
important factor in considering total damage associated
(Fig. 5, 6), where there were also concentric wrinkles
with the spot.