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Calonectn'a avesiculata
sp.nov.
l
T. S. SCHUBERT,
N.
E. EL-GHOLL, S. A. ALFIERI,
JR.,
AND
C. L. SCHOULTIES
Department of Agriculture and Consumer Services, Division of Plant Industry, P.
0.
Box
1269,
Gainesville,
FL
32602,
U.S.A.
Received September 19, 1988
SCHUBERT, T. S., EL-GHOLL, N. E., ALFIERI, S. A., JR., and SCHOULTIES, C. L. 1989.
Calonectria avesiculata
sp.nov. Can.
J. Bot.
67:
2414-2419.
Calonectria avesiculata
T. S. Schubert,
N.
E. El-Gholl, S. A. Alfieri Jr., et C. L. Schoulties sp.nov. is described from
leaf spots on
Ilex vomitoria
Ait. in Florida.
SCHUBERT, T. S., EL-GHOLL, N. E., ALFIERI, S. A., JR., et SCHOULTIES, C. L. 1989.
Calonectria avesiculata
sp.nov. Can.
J. Bot.
67
:
2414-2419.
Les auteurs dCcrivent le
Calonectria avesiculata
T. S. Schubert, N. E. El-Gholl, S. A. Alfieri Jr. et C. L. Schoulties
sp.nov. associC
i
des taches foliaires sur
l'Ilex vomitoria
Ait. en Floride. [Traduit par la revue]
Introduction
A severe leaf spot and twig dieback pathogen of
Ilex
vomitoria
Ait. was originally detected in southwest Georgia in
1967, and was described as
Cylindrocladium avesiculatum
D. Gill, Alf.
&
Sob. (1 I). The fungus is frequently encoun-
tered on propagating material, container-grown nursery stock,
and landscape plants in Florida, and can defoliate, seriously
debilitate, or kill its host (11, 13). In the fall of 1986,
C. avesiculatum
was diagnosed as causal agent of a severe
defoliation of an approximately 20-year-old planting of
I. vomitoria
in Gainesville, Florida. Many orange to red peri-
thecia were discovered on the diseased fallen leaves. Macro-
scopic and microscopic characteristics of the perithecia were
typical of
Calonectria
(22, 23), prompting an investigation
into the possible holomorphic relationship between the asco-
mycetous fungus and
~~li~drocladium av&iculatum,
for which
no sexual state has been reported. The purpose of this study
was to identify the perithecial fungus observed under natural
conditions and to produce under laboratory conditions the ana-
morph from the teleomorph, and vice versa.
Materials and methods
With sterile forceps, perithecia from the diseased fallen leaves
were picked and transferred to a drop of sterile deionized water
(SDW) on a flamed slide. To remove conidial and mycelial contami-
nation, perithecia were transferred through a series of individual
drops of SDW.
A
sterile cover slip was placed on top of the drop con-
taining the washed perithecia, and with pressure exerted against the
cover slip, separated asci, asci in groups, and free ascospores were
extruded. Asci and ascospores were washed off the slide with SDW
into an empty sterile Petri dish (100
x
15 mm). The suspension was
then dispensed onto 1.8
%
water agar (WA) and the excess was poured
off. These plates were incubated at 18-20°C for a period of 16-
20 h and examined under a dissecting microscope. Small squares of
the WA containing single germinating ascospores were cut with a
scalpel and were transferred to potato dextrose agar (PDA) sup-
plemented with
1
g/L KH,PO,. Eight single ascospores were selected
at random and were grown on
I. vomitoria
leaf segments on WA,
Arachis hypogaea
L. woody stem pieces (4-6 cm long) on WA, and
Dianthus caryophyllus
L. (carnation) leaf segments on WA (CLIWA)
to enhance sporulation (24).
Carnation leaves
(5
x
5 mm sections) were dried at 45-50°C for
'Contribution No. 626, Bureau of Plant Pathology, Gainesville,
Florida.
1 day. Properly dried leaf segments were green and crisp. Leaf seg-
ments were sterilized by propylene oxide fumigation (12). CL/WA
was prepared by placing several leaf pieces in a Petri dish (60
x
15 mm) and floating them on WA cooled to 50°C.
Cultures grown at room temperature (25
+
2°C) under ambient
laboratory lighting for 3 days were placed at 25
f
1 "C under fluores-
cent light (GE F20T12-CW) at approximately 3700 lx for 12
h
light
:
12 h dark intervals.
Results and discussion
Since cultures that were derived from single ascospores
produced more conidia on CLIWA than on
I. vomitoria
leaf
segments on WA or
A.
hypogaea
stem pieces on WA, CLIWA
was used throughout this study.
All single ascospore isolates produced conidia on CLIWA
(Fig. 1) and were identified as
C. avesiculatum
(1 I). Single
ascospore isolates did not produce perithecia in culture. The
eight single ascospore isolates were paired in all possible com-
binations on CLIWA and perithecia were formed in certain
cases within 4 weeks. The fungus appears to be a heterothallic
species. The isolates fell into two (Ala) sexual compatibility
groups, indicating bipolar sexual compatibility. Ascospores
from perithecia induced in vitro on CLIWA also yielded
C. avesiculatum.
Based on the average measurements, the perithecia pro-
duced in culture by the crossed single ascospore isolates were
larger than those formed on diseased fallen leaves (Table 1);
however, the color and shape of the perithecia were similar.
The dimensions of the asci and ascospores produced in culture
were nearly identical with those obtained from perithecia on
naturally infected fallen leaves of
I. vomitoria
(Table 1).
Ilex vomitoria
leaves showing spots were incubated in a
moist chamber at room temperature under ambient laboratory
lighting. Perithecia developed within 4 weeks primarily on the
lower leaf surface (Fig. 2).
Comparative measurements of perithecia (Fig. 3A), asci
(Figs. 3B and 3C), and ascospores (Fig. 3D) produced under
natural conditions and induced in vitro on CLIWA are shown
in Table 1. Ascospores that were obtained from perithecia
produced under natural conditions were 1-septate whereas
those obtained from perithecia induced on CLIWA were
mostly 1-septate. Of 226 ascospores that were observed from
induced perithecia, 90.7% were 1-septate, 4% were 2-septate,
and 5.3
%
were 3-septate.
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/
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SCHUBERT
ET
AL.
2415
TABLE
1. Comparative measurements of sexual structures of
Calonectria avesiculata
produced under natural conditions on diseased fallen leaves
of
Ilex vomitoria
and in vitro on carnation leaf segments on water agar
Ilex vomitoria
leaves (pm) Carnation leaf segments on water agar (pm)
No. No.
Structure measured Min. Mean Max. Min. Mean Max. measured Min. Mean Max. Min. Mean Max.
Perithecia
(H
x
D)"
32 348 418 516 318 405 487 61 437 577 715 353 470 566
Asci 94 82 131 189 13 20 32 44 97 128 187 14 20 25
Ascospores 338 22.3 41.1 74.3 3 5.4 7.4 226 18.8 44.6 79.2 3.5 5.2 6.9
FIG.
1.
Cylindrocladium avesiculatum.
(A) Conidiophore showing
an avesiculate stipe. x289.
(B)
1-septate conidium. ~462.
This represents the first report of a sexual state of
Cylin-
drocladium avesiculatum.
The teleomorph of
C. avesiculatum
is different from all known species of
Calonectria
with
Cylin-
drocladium
imperfect states (Table 2). Based on characteris-
tics of the anamorphic and teleomorphic states of these fungi
as outlined in Table 2, the fungus from
I. vomitoria
is consid-
ered morphologically distinct from previously described spe-
cies in this genus, and we propose the following new species.
Calonectria avesiculata
T. S. Schubert,
N.
E. El-Gholl, S. A.
Alfieri, Jr., et
C.
L. Schoulties sp.nov.
Perithecia superficialia in substrato, gesta singulatim aut in
turrnis parvis, aurantiaca usque ad rubra, pariete exteriore
aspero et verrucoso. In foliis
Zlicis vomitoriae
producta pro
parte maxima in inferiore pagina, (348 -)418(-516) pm alta,
(318-)405(-487) pm lata. Asci (82-)131(-189)
X
(13 -)20(-32) pm. Ascosporae granulosae, rectae usque ad fal-
catae, l-septatae, constrictae ad septum (22.3 -)41.1(74.3)
X
(3-)5.4(-7.4) pm. Perithecia inducta in partibus foliorum
FIG.
2. Perithecia of
Calonectria avesiculata
(as indicated by
arrows) produced in a moist chamber at room temperature on the
lower surface of a diseased
Ilex vomitoria
leaf. x6.6.
Dianthi caryophylli
super agarum aquosum. Perithecia
(437
-)577(-715) pm alta, (353 -)470(-566) pm lata; asci
(97
-)
128(- 187)
x
(14 -)20(-25) pm; ascosporae granu-
losae, rectae usque ad falcatae, pro parte maxima 1-septatae,
constrictae ad septum centrale (18.8 -)44.6(- 79 -2)
X
(3.5)5.2(-6.9) pm.
HABITAT:
In foliis
Zlicis vomitoriae.
LOCUS
HUIUS
HOLOTYPI:
Gainesville, Florida, U.S. A.
HOLCJTYPUS:
FLAS F55193, October 1986.
Calonectria avesiculata
T. S. Schubert,
N.
E. El-Gholl, S. A.
Alfieri Jr., et
C.
L. Schoulties sp.nov.
Perithecia superficial on substrate, borne singly or in small
groups, orange to red, with rough warted outer wall. On
Ilex
vomitoria
leaves, produced mostly on the lower leaf surface
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2416
CAN.
J.
BOT. VOL. 67, 1989
TABLE
2.
Comparison of 10 species of
Perithecia
(height
X
diameter, pm) Asci (pm)
Calonectria avesiculata
Orange to red 8-spored
348-516
X
318-487 82-189
X
12.5-32.4
Mean 418
X
405 Mean 131
X
20.2
Calonectria colhounii
Peerally Yellow 4-spored
247-463
X
309-515 (17) 104-156
X
13-18.2 (17)
Calonectria crotalanae
(Loos) Bell
&
Sobers,
comb. nov. Orange to red 8-spored
320-465
X
290-370 (5) 95-138
X
13- 19 (5)
313-475
X
286-405 95.2-138.7
X
13.6-19
Mean 382
x
348 (3) Mean 114.3
x
16.4(3)
280-450 diam. (19) 84.4- 135.2
x
12.6-20.4 (19)
Calonectria hederae
Booth
&
Murray
Calonectria ilicicola
Boedijn
&
Reitsma
Calonectria kyotensis
Terashita
Orange to red 8-spored
300-370
X
200-300 (7) 160-180
X
24-40 (7)
Orange 8-spored
400-500
X
320-370 (6) 110-140
X
16-18 (6)
415.8-724.7
X
332.6-570.2
Mean 544
x
433.9 (4)
Orange to reddish brown 8-spored
240-490
X
170-440 (28) 73-145
X
8-20 (28)
335-510
X
310-405 (25, 26) 83-145
X
14-22 (25, 26)
Calonectria quinqueseptata
Figueiredo
&
Namekata Orange to chestnut 8-spored
360-580
X
300-440 (10) 76- 126
X
13-22 (10)
Calonectria scoparia
Ribeiro
&
Matsuoka
Calonectria spathulata
El-Gholl et
al.
Orange to brownish red 8-spored
362.1-527.1
X
300.8-452.6 (21) 103.1-178.7
X
13.1 -25.7 (21)
Orange to red
318-536
X
273-457
Mean 422
x
367 (9)
4
-
8 ascospores
87-162
X
10-24
Mean 128
x
18 (9)
Calonectria theae
Loos Orange to red 8-spored
340-440 diam. (14) 99.2-145.5
x
16.3-25.8
273 -475
x
250 -475 Mean 122.6
x
21.3 (3)
Mean 374
x
371 (8)
248-481
X
227-382
Mean 374
x
313 (3)
"A.
Peerally (18) questions vesicle morphology of Boedijn and Reitsma's type strain (6). Number in parentheses indicates literature citation.
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SCHUBERT
ET
AL.
2417
Calonectria
with
Cylindrocladium
imperfect states
Ascospores (pm) Vesicles (pm) Phialides (pm) Conidia (pm)
1-septate Avesiculate tip, or, rarely, Reniform -doliform 1 -septate
22-74 x 3-7.4 very slight clavate 9-15
x
4.5-5.9 (11) 51-78
X
3.5-4.7 (11)
Mean 41
x
5.4 swelling (1 1)
3-septate Clavate
33.8-84.2
X
4.4-7.8 2-5.2 wide (17)
Mean 60
x
6.2 (17)
1- to 3-septate Globose
34-58 x 6.3-7.8 6- 13 diam. (5)
Mean 47.3
x
7.2 (5)
28.6-58.9
X
6.3-7.8
Mean 40.1
x
7.1 (3)
30.6-62.4
X
6.4-8 (19)
7.6-13.6 long (17) 3-septate,
rarely 1- or 2-septate
38.3-84.2
X
3.4-5.7 (17)
Reniform
-
doliform Mostly 3-septate
6.5-17
X
3.5-5 (5) 58-107
X
4.8-7.1 (5)
7.8-16.4 long (19) 58.5-107.4
x
4.7-7.1
Mean 78.9
x
5.9 (3)
60.4-105.2
X
4.8-7.2 (19)
3-septate Clavate or oval Doliform 3-septate
45-65
X
6-8(7) 30-40
X
8-10 (7) 12-15
X
5-6 (7) 60-80
X
6-8 (7)
6.4-14.4 wide (17) 44.2-102
X
5.6-9.2 (17)
1- to 3-septate Globose
38-57
x
5-7 (6) 5-8 diam. (6)
36.7-59.8
x
6.8-8.2 or clavate
Mean 49.6
x
7.4 (25) 5-11.2 (18)"
8-16
x
3-4 (6) Mostly 3-septate
10.2-13.6 long (18) 49-69
x
4.5-6 (6)
37.4-68
X
4-5.2 (18)
1-septate Globose Lageniform to doliform 1-septate
18-50
X
4-8 8.3- 17.9 diam. (27) 7-18
X
3-5 (28) 29-51
X
3-5
Mean 29
x
6 (28) 6-15.3 diam. (16) 8.3-15.2
x
4.1-7.6 (27) Mean 41
x
4 (28)
19.2-44.9
X
5.5-8.2 8-20
X
2.8-5.4 (16) 34-55
X
3.5-4.8 (27)
Mean 36.6x7.4 (25) 33-59 x 4-5.3 (16)
1- to 6-septate mostly Clavate
3-septate 12
x
2.5-3 (6)
30-80
X
4-7 (10)
1 -septate Ellipsoid to oval Doliform to oval 1-septate
27-65
X
4.1-7.8 (21) 17.5-32.7
X
5.2-9.2 6-10
X
3-4 (6) 40-50
X
3-4 (15)
Mean 26 x 6.7 (1) 7.7-16.3 x 3.2-5.4 (16) 32-60.1
X
3.3-5.5
19-37.7
x
8.3-14.5 Mean 50.7 x 4 (1)
Mean 29.6
x
11.9 (2) 34-57.1
x
3.5-4.8
19-22
x
8-10 (6) Mean 43.6 x 4.4 (2)
10-38
X
4-14 (16) 50-58
X
5-6 (6)
1- to 3-septate Spathulate Doliform 1- to 3-septate
Mostly 1-septate 11-38
x
3.5-12 9-17
x
4-6.4 39-86
x
4-6 (9)
14-71
x
3.2-7.4 Mean 20
X
7 (9) Mean 13
X
4.8 (9) Mean 58
X
5.1 (1 septum)
Mean 40
x
5.3 (9) Mean 66 x 5.5 (2 septa)
Mean 68
x
5.6 (3 septa)
1- to 3-septate Clavate Mostly reniform, Mostly 3-septate
42-70
x
5-8 13.6-28.7 x 4.1-8.2 (3) banana-shaped 62-100
x
3.5-6.5
Mean 57.9
x
7 (14) 7.7-16.5 x 3.5-5.3 (3) Mean 76.6
x
5.2 (14)
42.2-77.5
X
5.9-8.3 13-15.5
X
2.9-3 (14) 57.1-95.2
X
4.7-7.1
Mean 54.7
x
7.4
(3)
Mean 79.3 x 5.8 (3)
80
-
120
X
8 (20)
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CAN.
J.
BOT.
VOL.
67.
1989
FIG.
3.
Calonectria avesiculata.
(A) Perithecia exuding ascospores as indicated
by
arrows.
x
17. Perithecia were induced in vitro on CLIWA.
(B)
Whole asci. x520. (C) Asci containing eight ascospores. x578. Fig.
3D.
Ascospore showing constriction at septum.
x
1444.
(348-)418(-516) pm high, (318-)405(-487) pm wide.
Asci (82-)131(- 189)
x
(13 -)20(-32) pm. Ascospores
granular, straight to falcate, 1-septate, constricted at septum,
(22.3-)41.1(-74.3)
x
(3-)5.4(-7.4) pm. Perithecia
induced on carnation leaf segments on water agar. Perithecia
(437 -)577(-715) pm high; (353 -)470(-566) pm wide; asci
(97 -)128(- 187)
x
(14 -)20(-25) pm; ascospores granular,
straight to falcate, mostly 1-septate, constricted at the central
septum (18.8)44.6(-79.2)
X
(3.5-)5.2(-6.9) pm.
HABITAT:
Ilex
vomitoria
leaves.
TYPE
LOCALITY: Gainesville, Florida, U.S.A.
HOLOTYPE: FLAS F55 193, October 1986.
Two sexually compatible isolates derived from single asco-
spores were deposited in the American Type Culture Collec-
tion, 12301 Parklawn Drive, Rockville, Maryland 20852,
U.S.A. as ATCC 62947 and ATCC 62948.
Acknowledgements
The authors thank Judy M. Mattes, Jeffrey
W.
Lotz, and
John Corkery (Division of Plant Industry) for technical
assistance, and Dr. Lewis A. Sussman, Classics Department,
University of Florida, for rendering the Latin diagnosis.
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SCHUBERT
ET
AL.
2419
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