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PLAN'r PATHOGENIC SPECIES OF
STEt:iPHYLIUJ'.i \vALIJ�. IN NEW ZEALAND
A thesis presented in partial fulfilment of the requirements for the de�ree of
Doctor of Philosophy at hassey University
by
Gurmit Singh
June 1977
ACKNO\-ILEDG.EF ENTS
I am sincerely grateful to Dr. H.'r. \venham and Dr. K.S. Hilne for their advice and encouragement throughout the course of this study and in the preparation of this thesis.
I would also wish to thank:
Hr. D.H. Hopcroft of the Electron Hicroscope Laboratory, D.S.I.R. for his assistance with the transmission and scanning electron microgrCl.phs.
Hr. A.K. Hardacre of the PlCl.llt Physiology Division, D.S.I.R.
for use of climate rooms.
t-r. H.F. Heilson for technical assistance and valuable discussions.
The staff of the Central Photographic Unit, Massey University, for photographic processing.
Hrs. J. Parry for typing the thesis.
Dr. Ranjit Singh and Dr. Lalit Mehta for their constant encouragement and financial support during this study.
ABSTRACT INTRODUCTION CHAP.I'ER 1 .
CHAPI'ER 2.
CHAPrER 3.
CONTENTS
TAXONOhY OF ST.El•1PHYLIUE JiliD ASSOCIATED PLEOSPORA STATES
1 . 1 The Genus Stemphylium
1 . 1 . 1 Effect of Environmental Factors on Cultural and Morphological Features
1 . 1 . 2 Ornrunentation of Conidia and Conidiophores
1 . 1 . 3 Development of Conidia
1 . 2 The Genus Pleospora
1 .3 General Discussion
IDENTIFICATION OF NE'w' ZEALAND ISOLATES OF PLANT PATHOGENIC STfl1PHYLIUN SPECIES
2 . 1 Survey
2.2 Cross-PQthogenicity Studies
2. 3 General Discussion
SUPPID1ENTARY HYCOLOGY
3. 1 Ultrastructure of conidiogenesis in Stemphyliuru botryosum
3.2 Reversion of Conidiophores and Juvenile Conidia to Hyphal-like Growths
3 . 3 Spore Discharge Nechanism in Pleospora
iii
1 3 9
9 9 43
49 54 69
76
90 90 98
1 06 APPENDIX 1 A Previously Undescribed Chain-Forming Species 1 12
of Stemphylium
APPENDIX 2-5 Effect of Growth Hedia, Incubation Temperature, 1 15
Photoperiod and pH on Colony, Conidiophore and Conidiogenous Cell Features of Three Stemphylium
Isolates from Each of the Four Conidial Groups
APPENDIX 6 Composition and Preparation of Culture Nedia 1 32
REFERENCES 134
LIST OF T .J31ES Table
1 Stemphylium isolates, origin and conidial grouping 1 1
2a Effect of growth media on conidial features and 22 intensity of sporulation of three Stemphylium isolates
of conidial group 'A'
2b Effect of grO\vth media on conidial feu.tures and intensity 23 of sporulu.tion of three Ste�phylium isolates of
conidial Group 'B'
2c Effect of grov1th media on conidial features and 24 intensity of sporuL.ttion of three Stemphylium
isolates of conidial Group 'C1
2d Effect of growth media on conidial features and 25 intensity of sporulation of three Stemphylium isolates
of conidial group 'D'
3a Effect of incubation temperature on conidial features 26 and intensity of sporulation of three Stemphylium
isolates of conidial group 'A'
3b Effect of incubation telllperature on conidia.l features 27 Cilld intensity of sporulation of three Sternphylium
isolates of conidiu.l gr·oup 1 B 1
3c J::;ffect of incubation temperature on conidial features 28 and intensity of sporulation of three Stemphyliurn
isolates of conidial group 1C '
3d Effect of incubation temperature on conidial features 29
and intensity of sporulation of three Stemphylium isolates of conidial group 'D'
4a Effect of photoperiod on conidial features and intensity 30 of sporulation of three Stemphylium isolates of
conidial group 11\1
4b Effect of photoperiod on conidial features and intensity 31 of sporulation of three Stemphylium isolates of
conidial group 1B'
4c Effect of photoperiod on conidial features and intensity 32 of sporulation of three Stemphylium isolates of
conidial group 'C'
4d Effect of photoperiod on conidial features and intensity 33 of sporulation of three Stemphylium isolates of
conidial group 1C1
5a Effect of pH on conidial features and intensity of 34 sporulation of three Stemphylium isolates of conidial
group ' A '
Table 5b
5c
5d
6a
6b
6c
6d
7
8
Effect of pH on conidial features and intensity of sporulation of three Sternphyliurn isolates of conidial group 'B'
:Cffect of pH on conidial features and intensity of sporulation of three Stemphyliutn isolates of conidial group •c•
Effect of pH on conidial features and intensity of sporulation of three Stetnphylium isolates of conidial /.Sl'OUp 1D1
Effect of different teu�erature treatments on ascos
tromatu m;.�.tur...;.tion .<..nd 100rphologicu.l features of the ascostromu.ta, asci and ascospores of three Pleospora isolates of conidial croup 'A'
Effect of different temperature treatments on ascos
troroata maturC�.tion cmd morphologic;.;.l features of the ascostrom;.�.ta, asci and ascospores of three Pleospora isolates of conidial group 'B'
Effect of different temperature treatments on ascos
tromata maturation and morphological features of the uscostromata, asci and uscospores of three Pleospora isolates of conidial group 'C'
Effect of different temperature treatments on ascos
tromuta maturation and morphological features of· the u.scostromata, asci and ascospores of three Pleospora isolates of conidial group 1D'
Dimensions of the conidia, and ascospores of plant pathogenic Sternphylium species from different hosts examined in the survey
Results of cross-inoculation of 39 Stemphylium isolates on 9 different hosts.
V
35
37
59
60
62
78
86
Figure 1
2
3
4 5
6
7
8
9
LIST OF FIGURES
Effect of incubation temperature on conidial morphology of isolate Iv!U 9, conidial group 'A' (= Stemphylium botryosum)
Effect of incubation temper�ture on conidial morphology of isolate l"'iU 24, conidial group 'B 1 ( = Stemphylium elobuliferum)
Effect of incubation temperature on conidial morphology of isolate hU 2, conidial r;roup 'C' (= Stemphylium vesicariwn)
Effect of incubation temperature on conidial morphology of isolate NU 14, conidial group 'D'
Conidiophores and conidia of isolate HU 9 , conidial group 'A' (= Stemphylium botryosum), produced on 5%
V-8, pH 7.5, at 20C and in an 8h photoperiod
Conidiophores and conidia of isolate HU 24, conidial group 1B 1 (= Stemphylium globuliferum), produced• on 5%
V-8, pH 7.5, at 20C and in an 8h photoperiod
Conidiophores and conidia of isolate �1U 2, conidial group 'C' (= Stemphylium vesicarium), produced on 5%
V-8, pH 7.5, at 20C and in an Sh photoperiod
Conidiophores and conidia of isolate NU 14, conidial group 'D ' 1 produced on 5% V-8, pH 7.5, at 20C and in
an 8h photoperiod
Surface ornamentation of conidia and conidiophores of isolate l'·!U 9, conidial e;roup 1 A' ( = Stemphylium
botryoswn)
10 Surface ornamentation of conidia and conidiophores of isolate t-m 24, conidial group 1 B 1 ( = Stemphylium globuliferwn)
11
12 13 14 15
Surface ornamentation of conidia and conidiophores of isolate HU 2, conidial group 'C' (= Stemphylium
vesicariwn)
Surface ornamentation of conidia and conidiophores of isolate HU 14, conidial group 'D'
Conidium development of isolate MU 9, conidial group 'A' (= Stemphylium botryosum)
Conidium development of isolate NU 24, conidial group 1B1 (= Stemphylium globuliferum)
Conidium development of isolate �m 2, conidial group 1C1 (= Stemphylium vesicariwn)
16
17
19
39
40
41
47
48 50 51 52
Figure 1 6 17
18
19
20 21
22
23
24
25
26
Conidium development of isolat e l'-1U 14, conidial group 'D'
Asci and ascospores of isolate l'!U 9 , conidial group
'A' (= St emphylium botryosurn/Pleospora herbar um), produced on PCA �t 12C in d�rkness
Ji.sci and ascospores of isolate hU 24, conidial group 'B' (= St emphyliwn globuliferum/Pleospora sp.) produced on PCA and incubated at 12C in darkness
Asci cmd ascospores of isola.te HU 2, conidial gr oup 'C ' (= Stemphylium vesicar ium/Pleospora sp.) , produced on PCA and incubated at 12C in darkness
Asci and ascospores of isolate HU 14, conidial gr oup 'D', produced on PCA and incubated at 12C in darkness Conidia of St emphylium lot i, St et;,phylium sarcino.eforme and Stemphylium globuliferum pr oduced on
5%
V-8, pH 7.5, at 20C and in an Sh photoperiodConidia of St emphylium solani and Stemphylium lyco
g
ersici pr oduced on 5% V-o, pH 7.5, at 20C and in an h photoperiod
Sternphylium botryosum (isolate HU 80);
conidia by "blowing-out" of the ent ire genous cell
product ion of bulbous conidio-
C onidiophores and conidia of a Stemphylium sp. isolat ed from annual phlox. The fungus was grown on 5% V-8 , pH 7.5, and incubated at 20C in an 8h photoperiod Surface ornamentat ion of conidia and conidiophores of a St emphylium sp. isolate d from LJ.nnual phlox
Asci i:ind ascospores of a Sternphylium sp./Pleospora sp.
isolat ed from annu�l phlox. Cultured on PCA at 12C in darJr.ness
vii
53 65
66
68
71
72
79
80
81 82
27 Scanning electron micrographs of Stemphylium botryosmn 94 conidiogenous cells showing pore-like structures
28 Longit udinal section through the junction between 95 conidiogenous cell and conidiwn of Stemphylium botryosum 29 Scanning electron microe;raphs of Stemphylium botryosum 96
showing c onidium abstr ict ion
30 Scanning electron micrographs o f Stemphylium botryosum 96 after abstr ict ion of conidium
31 Conidiophor e prolifero.tion of Stemphylium botryosum 97 32 Stages in c onidiophore development of St emphylium 102
botryosum
Figure 33
34
35
36
37
39
40 41 42 43
Stages in vegetative reversion of Stemphylium botryosum conidiophores after exposure of cultures to 3h and 5h of l'Iylar-filtered light at 27C
Conidiophores of Stemphylium botryosum with vegetative regrov1th and aborted conidia after 5h exposure of cultures to t-'iylar-filtered light ut 27C
Vegetatiye regrO\�h of juvenile conidia of Stemphylium botryosum after exposure of cultures for v&rying length of time to l·1ylar-filtered light <J.t 27C
Vegetative regrowth of Stemphylium botryosum conidio
phores from the basal septum of conidiogenous cell and growing through a juvenile conidium
Transformation of hyphul-like regrowth of Stemphylium botryosmn conidiophores to functional conidiophores Nodular swellin5s of a Sternphylium botryosum conidio
phore induced by manipulation of photoperiod
Transformation of a Stemphyliwn botryosum conidium initial to a vegetative hypha induced by blue light ( 480 nm) at 2?C
Ascostroma and protruding ascus of Pleospora herbarurn
An overelongated ascus of Pleospora herbarum protruding from an ascostroma
A group of asci of Pleospora herbarum, some with outer
wall ruptured
Bitunicate ascus tip of Pleospora sp. (= Stemphylium vesicarium) showing the nipple-like invagination of the endoascus
102
103
103
104
104 105 105
110 110 111 111
44 An ascospore of Pleospora sp. (= Stemphylium vesicarium) 111 surrounded by a mucilac;inous sheath
45 Conidiophores and conidia of a Stemphylium sp. isolated 113 from hay straw. Produced on 5% v-8, pH 7.5, at 20C
v1i th an 8h photoperiod
1 ABSTHACT
A taxonomic study was conducted of 48 isolates of plant patho
genic Stemphylium Wallroth species, all of which produced a Pleos·pora Rabenhorst perfect state in culture. Specific identification was
attempted using gross colony characters and morphological features of the imperfect and perfect states. Only conidial and ascospore featur�s proved to be of taxonomic v�lue. The diagnostic conidial features were shape, dimensions, length/width ratio, number of longitudinal and lateral septa, number of lateral septal constrictions, ornamentations and pigmentation.
Ascospore features of taxonomic significance were dimensions, and shape of both juvenile and mature spores.
On the basis of these criteria it was concluded that three Stemphylium species were present, namely Stemphylium botryosum Wallroth, Stemphylium globulifermo (Vestergen ) Simmons, and Stemphylium vesicarium
(Wallroth) Simmons, each with a corresponding Pleospora state. This cross-pairing had value since identification of form-species allowed reliable prediction of the Pleospora species, and vice-versa.
The cultural conditions providing the best expression of
conidial features (and thus facilitating separation of Stemphylium species) were 5% V-8 agar, pH 7.5, 20C and an 8h photoperiod. Synchronous produc
tion of conidia was induced with an injury technique and ascostromata were matured most rapidly at either 12C or a diurnal temperature regime of 8h at 16C/16h at 8c.
A taxonomic survey of 1Go Stel�1ph;ylium isolates from 12 host species revealed the three previously mentioned species and a further undescribed species. The latter was isolated from annual phlox and was characterized by exceptionally large ascospores. s. vesicarium was by far the most common species, occurring on asparagus, chrysanthemum, blue lupin, Russell lupin, tree lupin, onion, pepper and tomato. The legumes were
hosts of more than one species; lucerne for instance was a host for .§.. botryosum, .§_. globuliferurn and _2. vesicarium, while Russell lupin and
tree lupin were only infected by �· botryosum and �· vesicarium. By contrast, lettuce and carnation were only infected by �· botryosum.
Cross-pathogenicity tests indicated that host specialization was relatively uncommon. �· botryosum from lettuce and the Stemphyliumsp.
from annual phlox were the only isolates exhibiting host specificity.
The results of ultrastructural studies of conidiogenesis, the
phenomenon of vegetative reversion of conidiophores and juvenile conidia, and the mechanism of ascospore release in Pleospora are discussed.
A previously undescribed, saprophytic, chain-forming species of Stemphylium is also described.
