Citation
Parasitism of Septoria Obesa Syd. and S. Chrysanthemella Sacc. on the cultivated chrysanthemum

Material Information

Title:
Parasitism of Septoria Obesa Syd. and S. Chrysanthemella Sacc. on the cultivated chrysanthemum
Creator:
Waddell, Henry Thomas, 1918-
Publication Date:
Language:
English
Physical Description:
vii, 82 leaves : illustrations ; 28 cm

Subjects

Subjects / Keywords:
Diseases ( jstor )
Fungi ( jstor )
Leaves ( jstor )
Lesions ( jstor )
Liquids ( jstor )
Mycology ( jstor )
Nitrogen ( jstor )
Pycnidia ( jstor )
Spore germination ( jstor )
Water temperature ( jstor )
Chrysanthemums -- Diseases and pests ( fast )
Septoria ( fast )
Genre:
bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

Notes

Bibliography:
Includes bibliographical references (leaves 79-81).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Henry Thomas Waddell.

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University of Florida
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University of Florida
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This item is presumed in the public domain according to the terms of the Retrospective Dissertation Scanning (RDS) policy, which may be viewed at http://ufdc.ufl.edu/AA00007596/00001. The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
Resource Identifier:
37973898 ( OCLC )
ocm37973898
Classification:
LD1780 1959 .W116 ( lcc )

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Parasitism of Septoria obesa Syd. and S. chrysanthemella

Sacc. on the Cultivated Chrysanthemum













By
HENRY THOMAS WADDELL


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF *PHILOSOPHY













UNIVERSITY OF FLORIDA
August, 1959















ACKN0IZDGINT


The writer greatly appreciates the guidance, helpful suggestions, and aid In manuscript preparation given to him by Dr, George F. Weber, chairman of his supervisory committee. Appreciation is expressed to the other members of the supervisory cosmitteeDr. Mildred Griffiths Dr. Pharos Decker, Dr. P. H. Senn,, and Prof, Albert Muller-for their help and consideration,

Acknowledgments are gratefully made to the Uidversity of Florida Graduate Council, the Southern Fellowships Fund, and the National Science Foundation for fellowships without which this work could not have been completed.

Sincere gratitude is expressed to Myra, the devoted wife of the writer, for encouragement and for the performance of Innumerable tasks.-including the typing of this manuscript.
















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q -< j4













TABLE OF CON78NTS

Page

ACKNOWLEDWENTSee as es eas a . ...... .e so Ii

LIST OF TABIES . * * * * * * * * * . . * . * a . * * * * * * iv

LIST OF FIGURES . . . . . . . , . . . . . . . . . . . . vi

INTRMDIKTION - & . 0 * * * * * * & * * * a * * * * * * * * 1



Host Range . . . . . . . . . . . . . . . . . . . . . . . 2
Geographical Distribution . . . . . a......... 3
Economic Importance 9. . . . *. * a...... 4


Tympnomy . . . . . . . . . . . . . . . . . . . . . . . . 12


morphology . * * * * * * . 0 . . . . . . . . . * . . . . 16
Physiology . . . . . . . . . . . . . . . . . . . . . . . 36
Pathogenicity * . * . . . a . . . . 9 . . . . . . . a . 57



Seasonal Development of the Disease * * * **. 66
Sources ofthe noculnn ,, . .... .... 66
Spore Dissemination ..........a........a 0 0 68
Climatic Conditions Affecting Infection and Disease
Development . & . . . . . . . . . . . . . . . . . a * 69
Host Penetration a . . . .. . . .. * 000 * 70
PathoclogAnamyto. . . .........a.. 72

SUMMAR . . 0 0 a 0 0 0 0 . 0 4 0 a 0 * 0 . 77

MIERATUE CI8D ** * * * ag e * a a a cc.a.9a 9aaa 79


Ii












LIST OF TABLES


Table Page

1. Morphological Characters of Septoria obosa as
Reported by Different Investigators ......... 16

2. Morphological Characters of Ro.2toria chrysanthemella
as Reported by Different Investigators . . 0 . . . . 17

3. Dimensions and Number of Septa of Pyonidiospores, and
Diameters of Pycnidia of Septoria obesa. Specimens
Collected at Various Locations in the Unaited States * 29

4. Dimensions and Number of Septa of Pycnidiospores and
Diameters of Pycnidia of Septoria chrysanthemella.
Specimens Collected at Various Locations in the United


5. Comparison of Pycnidiospore Dimensions on Six Chrysanthemum Varieties Inoculated with Isolate No. 1 of
Septoria obesa. * 0 0 00 00 * * e ag...... 31

6. Comparison of Pycnidiospore Dimensions on Six Chrysanthmum Varieties Inoculated with Isolate No. 1 of


7. Comparison of.Septoria obesa. and Septoria chrysanthemella. Specimens Collected in the United States:
Pycnidiospores and Pycnidia , , , , , . * 0 0 * 0 0 0 34

8. The Effect of Hydrogen-ion Concentration on the Growth
of Septoria obesa in a Synthetic Liquid Medium . . . 40

9., The Effect of Hydrogen-ion Concentration oft the Growth
of Septoria chrysanthemella. In a Synthetic Liquid


10. The Effect of Temperature on the Growth of Septoria.
obesa in Synthetic Liquid Media . 0 0 a 0 * 0 a 0 . 42

ill The Effect of Temperature on the Growth of Septoria obesa. on Semi-synthetic Agar Medium , , * . . 0 0 0 43

12. The Effect of Temperature on the Growth of Septoria cRyUsanthemella in Synthetic Liquid Medium . . . . . 44


iv










LIST OF TAKlES (continued)


Table Page

13. The Effect of Temperature on the Growth of Septoria chrysanthemella, on Semi-synthetic Agar Medium , 45

14. The Effect of Carbon Source on the Growth of Septoria. obesa, on Semi-synthetic Agar Medium . . . . 47 15. 7he Effect of Carbon Source on the Growth of Septoria chrysanthemella, on Semi-synthetic Agar Medium 48 16. The Effect of Nitrogen Source on the Growth of Septoria, obesa. on Semi-synthetic Agar Medium ...* 49

17. The Effect of Nitrogen Source on the Growth of Septoria chrysanthemella, on Semi-synthetic Agar


18. Percentage of Germination of Pycnidiospores on Water Agar at Various Temperatures . . . . . . . * . . . . 54

19. Severity of Disease on Mature Plants of Fifteen
Chrysanthemum Varieties Inoculated with Septoria obesa,
in thelField .* e * ae g. .. e o* *o...e* 61

20., Severity of Disease on Twenty-two Chrysanthemum
Varieties Inoculated with Septoria obesa, In the Green21. Severity of Disease on Twenty-two Chrysanthemum
Varieties Inoculated with Septoria chrysanthemella,
In the Greenhouse * *. . . . * * a * 9 .***...9a* 63

22., Severity of Disease on Fourteen Chrysanthemum
Varieties Inoculated with Septoria chrysanthemella,
in the Field * e & o * * * * * * 9 * 9 @ * * * e * e 64


v












LIST OF FIGURES


Figure Page

1. Lesions on old Chrysanthemum Leaves 6 Weeks after
Inoculation with Spores of Soptoria obesa, * a . e * * e e 8

2. Lesions on Young Chrysanthemum Leaves 6 Weeks after
Inoculation with Spores of Septoria obesa, . a * & . e * * 9

3. Lesions on Old Chrysanthemum Leaves 6 Weeks after
Inoculation with Spores of Septoria chrysanthemella . ,. * 10

4. Lesions on Young Chrysanthemum Leaves 8 Weeks after
Inoculation with Spores of Septoria chrysauthemella . , . 11

5. Longitudinal Section of a Pycnidium of Septoria obesa
in Chrysanthemum Leaf Tissues . . . . . . 21

6. Pycnidiospos of Septoriaobesa s e .. * . 22

7, Pycnidia. of Septoria obesa, on Potato-Dextrose-Agar

8. Longitudinal Section of a Pycnidium of Septoria chrysanthemella, in Chrysanthemum Leaf Tissues . . a * . . . a 25

9. Pycnidiospores of Septoria chrysauthemella . . . * e * 26

10. Pycnidia. of Septoria. chrysanthemella, on Potato-DextroseAgar Medium . 27

11. Camera Lucida Drawing of Pycnidiospores of Septoria
chrysanthemella and Septoria obesa. . . . . . . . . . . . 35

12. Growth of Septoria obesa. and Septoria. chrysanthemella.
on Semi-synthetic Agar Medium at Optimum Temperatures
at theBud of 4Days ........ ...*0000 46

13. Growth Characteristics of Septoria chrysanthemella on
Semi-synthetic Agar Medium Supplied with Different Nitrogen
Sources (1) L-asparagine, (2) L-arginine,, (3) glycine (4)
potassium nitrate, (5) azmionium sulfate,, (6) L-leucine,,
(7) minus nitrogen . . . . .. . ... . . . . ... . . 51


vi










LIST OF FIGURES (continued)


Figure Page

14. Pycnidiospores of Septoria chrysanthemella GerminatIng on Solid Medium . . . . . . . . . . . . a . . . . 53

15. Pycnidiospores of Septoria chrsanthmella GermInatIng inWater a .. 0 0 .. 0&0 06a 0 *0 0 a* 53

16, Stomatal Penetration by a Hypha from a Germinating
Pycnidospore of Peptoriaobesa . . . . . . . . &0 73

17, Stomatal Penetration by a Hypha from a Germinating
PycnidiLospore of Sept~ria chrsanthenella . . . . . . 73 18. Cross-section of Host Leaf Showing Intercellular
Invasion of 11esophyll Tissue by Septoria obesa


19. Cross-section through a Leaf Lesion Showing PycnidIa
of.Sepiaora . 0 .. 0 0 0 0 0 0 0 76

20. Cross-section through a Leaf Lesion Showing Pycnidia
of.Septoria cha yseela . , , , 0 * ........... 76


vii














INTMUCXTION


Two distinct septoria diseases of the cultivated chrysantheum commuonly occur in the United States and in several other parts of the world where the host Is grown,

The development of similar disease symptoms has led to uncertainty and error. Confusion abounds with respect to the identity of the causal organism, Information Is Incomplete or lacking on the morphology and physiology of the causal parasites and also on their relative pathogenicity on various varieties of the cultivated chrysanthemum, Various host-parasite relationships are not known, especially the mode of penetration by the fungus and pathological anatomy.

These investigations were inaugurated to explore those areas where information is Incomplete or lacking with respect to-the diseases,, the pathogens,, and the host-pathogen relations.

The results are reported herewith* The experimental. work is supplemented by a review of the literature in all areas pertaining to the diseases$ the pathogens,* and the host-pathogen relations.


I














THE DISEASES


The causal organisms of the diseases are Septoria obesa and Septoria chrysanthemella. Various common names such as leaf spot, black spot, brown spot,, septoria leaf spot, leaf blight, leaf scorch, and leaf blotch have been used to describe the diseases. Each of the fungi has been referred to under most of these common names, Weiss

(39) refers to the disease caused by. obesa as leaf blotch and to the disease caused by. chrysanthemella as leafspot.




The disease caused by Septoria obesa, was described on Chrysanthezum articum by H, and P. Sydow (36) and later reported on hsn themum. morifolium by Heummi and Nakamura (22),* Various investigators have since found it to be widespread on C., morifolium.

The disease caused by Septoria chrysanthemella. was described in 1892 by Cavara (32) on.Chrysanthemum morifolium. Since that time it has been reported on this host by numerous investigators in many parts of the world. The disease has also been reported on Chrysanthemum cinerariaefolum (23) and on Chrysanthemum maximum (14)0 However, crossinoculation tests have not been made, and# consequently,, proof that the diseases are identical is lacking*


2






3


Geographical Distribution


As previously noted, the two diseases have probably often been confused, Consequently, reports on geographical distribution may not be entirely correct,

The disease caused by Septoria obesa was reported by Rerni and Nakamura (22) to occur In Japan, Formosa,, and the United States, Loos

(24) described the disease in Ceylon in 1941. According to Weiss (39) It has been reported in Connecticut, Maryland, New Jersey, New York, Texas, and Washington but Is probably much more widespread. In addition to some of the states listed above by Weiss, the writer has collected the disease specimens on the leaves of the host in Florida, Indiana.. Illinois, Ohio, South Carolina, North Carolina, Iowa, Missouri, and Tennessee,

The disease caused by Septoria chrysanthemella has been reported onChrysanthemum morifolium in China (22), Japan (22), Formosa (22), Denmark (33),, France (5), the Azores (2), Germany (30), Belgium (26), Czech6slovakip. (28), England (35), Mozamubique (8), Iceland (7), Italy

(32), Costa Rica (27), and New Zealand (4)o In the United States the disease was noted by Halste~.d (20, 21) in Now Jersey as early as 1891 and was noted by Beach (1) In New York in 1893. According to Weiss

(39) the disease occurs throughout the eastern and central states to Florida and Texas and also occurs in Colorado and California.,

The writer's investigation strongly Indicates that the disease caused by Septoria obesa is much more prevalent in the United States than is the disease caused by Septoria chrysanthatuella. Specimens of diseased chrysanthemum leaves from 13 southern, eastern, and central






4


states were examined. The Septoria obesa disease was found on collections from 13 of these states, whereas the Setr chrysantheanella

disease was found on specimens only from Iowa, Texas,, and Tennessee.


Economic Importance

The chrysanthemum is one of the most Important flowers of

commerce in the United States and also In several other parts of the world, Most of the plants are ultimately sold as cut flowers, but there is also a large trade in potted plants and in rooted cuttings for plant-Ing in private gardens,

The septoria leafspot diseases were very severe in commercial beds in the United States until World War II. The diseases are now controlled to a great extent in commercial plantings by dithiocarbamate . sprays but frequently become seer during extended rainy periods. The v diseases remain a problem in private gardens where control measures are usually less rigidly practiced.

The writer agrees with the statements of Dimock (10) concerning losses due to septoria leaf spots:

1, Spotting of foliage seriously detracts from the
quality and salability of cut flowers.

2, Even if spotting is confined to the lower leaves,
the loss of lower foliage may seriously reduce both
the quantity and quality of blooms.

As previously stated, the disease caused by Septoria obesa Is

apparently more common than the Septoi chrysanthemella disease In the United States, and, as will be described later, it is considerably more severe.






5


The following descriptions a"e based an observations of the

natural development of the diseases In the field and greenhouse and on inoculation experiments under various controlled conditions.

MESCRIPTIONS,-The disease caused by Septoria obesa is usually considerably more severe than the disease caused by A!Sepor chrysanthemella. Lesions begin as small chiorotic areas which usually become dark brown or black and enlarge to form circular to very irregular areas with distinct margins* Frequentlys however, the young lesion develops into a blackish or brownish blotch-like lesion of indefinite size and shape with Indistinct margin. The latter type of lesion becomes black and the margin becomes distinct during rainy weather. A yellow halo usually surrounds the lesion, A single lesion may spread to occupy 1/3 or more of a leaf,, and a leaf may be killed by one or two lesions. The lesions are distinct on both surfaces of the leaf but are darker on the adaxial surface. Pycnidia are usually abundant on both surfaces of the lesions,

The disease attacks and kills the lower leaves first and under favorable conditions progresses upward& finally attacking the upper leaves and the involucral bracts, Entire shoots or entire plants are frequently killed* The symptoms are shown in Figsb I and 2.,

The disease caused by Septoria chrysanthemella occurs on

susceptible chrysanthemum plants of all ages* The first symptom Is the appearance of very small chiorotic spots, barely discernible without unification, on the leaf blades* Under favorable moisture and humidity






6


conditions, these spots become black in a few days and gradually enlarge to form black, circular or elliptical spots, mostly 1 cm or less in diameter but sometimes as large as 2 cm in diameter. These lesions usuealy show distinct margins, and they are sometimes surrounded by narrow chiorotic halos. They are visible on both surfaces of the leaf but are considerably more conspicuous on the adaxial surface.

When the adaxial surf ace of a lesion is examined under a hand lens, numerous black pycnidia are seen embedded in the leaf tissue. There may be only a few lesions on an infected leaf, or there may be so many that they coalesce, thus forming large necrotic areas, The centers of old lesions sometimes become grayish in color,

The lower leaves of a plant are infected first, and if Infection is severe they die and hang downward on the stem. Following periods of rainfall, the disease progresses upward on the plant, and the uppermost leaves and the involucral bracts may eventually become infected, Symptoms of this disease are shown in Figs. 3 and 4.

COKARI84RI-Advanced symptoms produced by Septoria obesa, under favorable conditions of moisture and temperature are distinctly different from those produced by Septoria chrysanthemella, the leaf lesions being large and circular to irregular in shape. S, chrysanthemella causes circular to elliptical leaf lesions which usually do not exceed

1 cm In diameter, but these lesions may coalesce to form black, irregularshaped, necrotic areas; however, when closely observed,, the individual spots making up the irregular areas are easily discerned, Another point of difference Is the occurrence of abundant pycnidia on the abaxLa]l surface of S, obesa. lesions, whereas S, chrysanthemella lesions either







7



lack or have only a few pycuidia on their abaxial surfaces&

Young lesions produced by Septoria obesa are frequently small and circilar,, thus resembling those of.Septoria chrysanthewella. The two diseases can be distinguished with certainty by-observing the pycnidiospoa'es produced in the lesions* The morphology of the pycnidiLospores will be described later,
























AP


Fig, 1.-Lesions on old claysanthmunm leaves
6 weeks after inoculation with spores of Septoria obesa (X 1/3).


NEI






0


I7


ALL


J.


4g~ C-


Fig. 2.--Lesions oun young chrysanthemum leaves 6 weeks after inoculation with spores of Septoria obesa, (X 3/4),


I


Ats


YWWI'





























L


Fig. 3.-eso 2 3.ailhimmu leaves
6 weeks after iou..0rp.esof Septori a chrysantliemella -." X e3


20




















im.


I


d.~ '.~I


IA
*


Fig. 4.--Lesions on y N~eeks af ter inoculdtion Y ,vsanthemella (X 3/4)o


c'C snthemnum leaves with spores of Septoria


11


I


u















13C&LUSAIJ -OBNInsM




Th. Identity of the numerous species of Sqptoria Is based on morphological differences,, physiological adaptations, and specificity for different host plants. Regardless of morphological similarities, Septoria app, specific for different, not-closely-related hosts a"e placed In separate species. Morphologically similar septorias which ame specific for different but closely-related hosts, such as hosts In the same genus or family, are usually placed In different species but are sometimes given varietal names In the ame, species.

In discussing the taxonomy of the two causal organisms under consideration, septorias described on different host plants will be considered to be of different species unless proved Identical by cross-inoculation experiments,

WNTOMI OH~TSAN1I3IrnJA SACC-In 1892 Cavara (32) described a fungus causing spots on chrysanthmm leaves In Italy., He gave this fungus the binomial Septoria chrysanthemum Cave However, since Allescher (32) had used this name In 1891 In describing a very similar fungus an the leaves of Chrysanthemum leucanthemum In Bavaria, Saccardo

(32), in 1895, changed the name of Cavara' a fungus to Septoria chysnthemella Caye In 1907 Nagnus (25) stated that the latter fungus should


12







13


be referred to under the name of Septoria chaysanthemlla. Sacco# as the name had been changed by Baccardo and not by Cavara.

In 1897 Rostrup, (33) In Denmarks described a fungus parasitic on the chrysanthemum under the name of Septorla chrysanthahi Rostre Due to the presence of Allescher 's species * Saccardo and Sydow (33) changed the nme to Aggtor rotui Sacc. and Syd. In 1899. Magnus

(25) and Heml and Nakamura (22) state that this fungus Is the same as Septoria chrysanthemella Sacc. After studying the descriptions of the two fungi * this writer agrees with them.

In 1907 in Dohaiia,, Bubak and Maat (34) described a fungus attacking the chrysanthmn under the name of qtr chrysanthemiindici Bub, & Rob. iagnus. (25) believed this fungus to be synonymous with fttr chrysauthemella Baca, If the original description is

accurate the opinion of Kagnus Is incorrect, for the pyonidiospores, of S. chrysanthemella are filiform and 0-5 septate# ubereas Bubak and Kabat described the pycuidiospores of their fungus as being tapered from the base to the apa and 10-15 septate. According to this description the pyenidiospores of the latter fungus are similar to those of Septoria obes Syd. When the type specimen of Dubak and Kabat was examined by Komi (22)0* he found that the pyonidiospores waenot as described,, being more similar to those of S. chrysanthemella Saco Since more than one species of Septoria may occur on the same plant, the synonymy of 8, chrysanthemi-indici with either 8. ohrysnthmlla. Saco or S. obesa Sydo is questionable,

According to Chiff lot (5) !Itr varians Joffrin Is a synonym of Septoria chrsanthamella, The writer has not had an opportunity






14


to read Joffrin s description of this fungus$* but an described by Chifflot the pyonidiospores aro filform and many-cealed and figures of the spores show then to be obolavate and 10-14 septate. If Chlff lot's description Is accurate, Septoria varians Is not the same as Septoria chrsanthemilla.

The valid name and synonyms of the fungus under consideration are as follows:

Setr ohrysenthmella Saco. (sub nown. Septoria
chrysanthemells Cay. 1895)

synonyms:
Septoria chyanh Cay. 1892.
Setr chrysantheml Rostr. 1897.
Setr rotuAi Sacm, & Syd. 1899.


SP1TOA ONA SYD-.In 1914 H, and P. Sydow (36) described a new fungus, Setr a besa, Syd. * parasitic on, rsatm arcticwa,

a wild plant of northern Japan*

In 1917 HiQii (22) found two different septorias parasitic an Chrysanthmnm morifolium in Japan, One species was identified as Septo#i chrysanthemella. The other species was first Identified as Septoria chrysanthemi-indici Bub. & lab. * but after examining the type specimen of this fungus,, Hoodi reversed his opinion, Subsequently Komi studied Sydow's description of ikeptoria obesa, and examined the type specimen. He found that his species was morphologically Identical to Egor obesa Syd. and stated that the two are synonymous. Theoretically * Hwmid's species should have been described as a new species or as a variety of B., obesa. The two occurred on different hosts and cross-inoculation experiments were not conducted.






15


In 1893 1lis and Dearness (15) In Canada described a fungus parasitic on chrysanthemums and named It Cylindz'osporiu chrysanthemi Be & Do The spores of this fungus weevery similar to those of Septoria oboe& Syd, and the spore-bearing structures were similar In shape and aso to pycnldIa of 8. obea, Sydobut lacked a pycaIdIa1 wail. The spore-bearing organ was therefore considered to be an acervulus and not a pycaidium. Trent (37) ogggests that this fungus Is synonymous with Septori a rsanthowella Baca., and Greene (18) states that It may be the sam~e as Leptoria obea Syd. Unless the original description can be shown to be wrong, the writer cannot consider this fungus to be classified In the genus eptorl.

Weiss (39) lists Setr leucanthami Saco, & Spec. Septoria marspra Dearn. * and Septoria cercosporoldes frail as probable synonyms of #qtr obese, Sacco The writer cannot agree that any one

Of these three fungi Is synonymous with S. obe& Sacc.*p as all three were described on Chrysanthemum leucanthm and cross-inoculation

e~zeraetshave not benconducted, IFurhroe the pycnidiospores, of both Septoria leucanthemm (31) and septoria WMwcoporia (18) are filiform. This differs from the pycnidiospores of SeW obese, which

are obalavate. The pycnidiospores of P~taria cercosporoides as given by Cooke (6) after Trail appear to be very similar to those of S. obesa but are somewhat smaller,

The valid name of the fungus under consideration is:

SeptorI obesa Syd. 1914.
Synonyms: none.







16


Morphology


Both Septoria obesa d~ad Septoria citrysanthemella produce

mycelia, pycnidia, and pycnidiospores in leaf tissues of susceptible hosts and in suitable artificial culture media. A perfect stage of either of these fungi has not been reported. Voglino (38) in 1901 reported what he termed a phoma stage of S. chrysantheinella on the leaves and stems of chrysanthemums. This stage has not been observed by any other investigator, and it seems likely that it was produced by a fungus distinct from S, chrysanthemella.

Several authors have given descriptions of the pyenidia and pycnidiospores of Septoria obesa. end Septoria chrysanthemella. A summary of these descriptions is given in Tables 1 and 2.


TABLE 1

NMOHOGICAL CHA.RACTERS OF SEP70RIA OMSA AS RE~PORTED BY DIPMERENT INVESTIGATORS



Investigators
Morphological H, & P, Hemmi &
Character Sydow (36) Nakamura (22) Loos (24)


Diameter of pycnidia 120-160 U 75-200 u 87-173 u

Length of pycnidiospores 50-100,* rarely 41-124 u 66-87 u
120 u

Width of pycnidiospores 3-4,5 u 2.5-4.4 u 3.2-3.6 u

Number of septa 5-12 4-12 up to 9

Shape of pycnidiospores narrowly whip-shaped Rounded at
obelavate base, tapered
toward apex






17


TABLE 2

MORPHOLOGICAL CHARACTERS OF SEPTORIA CHRYSANTJEJIELLA
AS REPORTED BY DIFFERENT INVESTIGATORS


Investigators
Morphological Heanmd &
Character Cavara (32) Bostrup (33) Nakamura (22)


Diameter of pycnidia 100-120 u 53-148 u

Length of pycnidiospores 55-65 u 40-50 u 23-72 u

Width of pycnidiospores 1,5-2 u 2 u 1.2-3 u

Number of septa 0 0-5

Shape of- si{~~e filiform filiform filiform
I \


In the summer of 1958 diseased chrysanthemum leaves were obtained f rom various parts of the United States, Of the 16 leaf specimens found to be infected with Setr 12 were infected with Septoria obesa and

4 with Septoria chrysanthemella. Only 8 of the specimens infected with S. obesa were used in this and other studies in this paper; all 4 S, chr~vsanthemella samples were used, The geographical sources, specimen numbers, and the numbers assigned Isolations are given below: Septoria obesa


Specimen and Isolation No,
1
2 3
4
5 6 7
8


Geographical Source
Gainesville,, Florida Ithaca, New York Bloomington, Indiana Urbana, Illinois Ashtabula, Ohio Bel Air, Maryland Spartanburg,, South Carolina Ames, Iowa







is


Septori achrysanthemel la

Specimen and
Isolation No. Geographical Source
1 Troy,, Tennessee
2 Beaumont, Ta1xas
3 Ames, Iowa*
4 Ames, Iowa*

*Specimens from Ames came froim different nurseries.

Studies were made of the morphological characters of the fungi in the original leaf sams, in pure culture,, and in the leaves of several chrysanthemum varieties artificially inoculated with pure cultures of the pathogens,

IODS ANID MAElRIALS.--ln the observations of pycnidiospores

in the original leaf specimens, randomization of the spores was obtained by soaking several lesions of each specimen in water for three hours and then stirring the resulting spore suspensions vigorously, In order to stain the septa of the pycaidiospores, an iodine solution was added to the suspensions, Drops of the suspensions were then placed on glass slides and pycuidiospores measured by. aze of a previously calibrated ocular micrometer. Septoria obesa pycnidiospores were measured for width near their bases, whereas those of Septoria chrysanthemella were measured for width midway between their bases and apices. Lengths and widths were measured at 440 and 970 magnifications,, respectively. A mechanical stage was used to insure that no spore was measured more than once.

The procedure used in securing isolations from the original leaf specimens was as follows: The diseased leaves were soaked in






19


1:1000 mercuric chloride for 2 minutes and then for 5 minutes each in 3 changes of sterile distilled waters The diseased leaves were then

placed on moist filter paper in sterile petri dishes and maintained for 24 hours In a constant temperature chamber at 80C. At the end of this period pyonidiospores in gelatinous matrices had oozed from many of the pycnidia. With the aid of a stereoscopic microscope, spore suspensions were prepared by touching the spore masses with the tip of a sterile needle and rinsing the needle tip in sterile, distilled water, The suspensions were then plated on water agar in petri dishes and incubated for about 43 hours at 200C, Bacterial contaminants had formed colonies by the end of this period and were easily avoided. Germinated spores were removed singly with a hooked needle and transferred to potato-dextrose-agar medium in petri dishes.

Measurements of pycnidiospores obtained from several chrysanthemum varieties previously artificially inoculated with isolate No, 1 of Septoria chrysanthemella, or with isolate No, 1 of Septoria obesa were made by using the procedures described earlier in this section. The artificially inoculated material was from an experiment concerned primarily with pathogenicity, and an account of the techniques employed is given under that topic.

Studies of morphological characters of the fungi in host leaves and in culture on potato-dextrose-agar medium were made from serial sections prepared by the paraffin method and examined unstained or after being stained with safranin, safranin-fast green, cotton-blue, or iron hematoxylin,






20


]RESULTS--The following descriptions of the morphological characters of the two species of Septoria under consideration are based on the studies described above,

Septoria obesa,--Pycnidia formed in host tissues are typically globose or subglobose but sometimes pyriform or irregular in shape, 60-160 microns in diameter, Short to rather elongate beaks are present, and the diameters of ostioles vary from almost no opening to that measuring one-half the diameter of the pycnidium. A pycnidium of S. obesa Is shown in the leaf tissues of the chrysantheum In Fig. 5. Pycni dial walls are composed of 3 to 7 layers of cells which are isodiametric to elongate in shape, The inner layer of cells is hyaline, and the outer layers are brown. Conidiophores are composed of cells of the inner wall of the pycnidium which have become slightly elongated; they are hyaline, 3-7 microns in length, The mycelium is mostly 3 microns or less but sometimes up to 6 microns in diameter, finely granular, hyaline, constricted at the septa, obtusely branched,, and septate at the points of branching., A mature pycnidiospore of S. obesa is relatively large and rounded at the base and tapers from the basal end or from near the mid-region of the spore to the apex (Fig, 6). In culture on potato-dextrose-agar medium the morphology of pycnidiospores is similar to that described above, Pycnidia vary greatly In shape, often forming extensive flattened cavities, and the ostioles are not well defined (Fig. 7). Most of the mycelium is devoted to producing spherical or elliptical-shaped cells which go into the formation of pycni di al walls.a






21


~~51









Septoria obesa in chrysanthemum leaf tissues (X 476),






22


___________1/
I





4
4






Fig. G.--Pycnidiospores of Septoria obesa (X 476).






23


40J

















Fig, 7.--Pycnidia of Septoria obesa on potatodeoxtrose-agar medium (X 111),





24


Septoria chrysanthemella.-Pycnidia. formed In host tissues

(Fig. 8) are mostly pyriform but frequently globose or subglobose in shape, 40-124 microns In diameter, and slightly to 1/2 brumpent, Ostioles occur at the ends of short to rather elongate beaks and vary in diameter from being almost closed to being a-nut 1/2 or more the diameter of the pycnidium when spores are being exuded, The walls of the pycuidia. are composed of 3 to 6 layers of compact cells which are usually nearly isodiametric but sometimes 3 or 4 times as long as broad. The outer cell layers are brown in color but the innermost layer is hyaline, Conidiophores are composed of the inner cells of the pyonidial wall which have elongated slightly toward the center of the pycnidium. They are hyaline and 3 to 6 microns In length. PyonidiLospores (Fig, BY) are filiform,, usually straight but sometimes slightly curved, uniform in width and bluntly pointed at base and apex,, obscurely 0-5 septate, 22-70 x 1.8-3 microns, hyaline, finely granular, anel usually several guttulate. The mytelium Is similar to that described above for Septoria obesa.

In culture on potato-dextrose-agar medium, the morphology of the pycnicliospores. of Septoria chrysanthemella is as described above. Pycnidia in culture are globose to very irregular in shape and the walls are usually much thicker than in host tissue, The first pycnidia formed are Elmost superficial on the surface of the culture and are oriented with their ostioles communicating with the internal portion of the culture (Fig, 10), On(ly occasionally do ostioles open on the surface. Pycnidia formed in deeper layers of the culture are oriented variously. The morphology of the mycelium is similar to that described for Septoria obesa.






25


v2


AWWI&#





-4"


I


Fig, . BLongitudinal section of a pycnicliun of Sptoria. chrysanthemnella in chrysanthemum leaf


I


IE!rO






2C


~'Of






AlW



ig.9Peiisoe o etr hyate
malla X 476)




















-77




































w~potatepdetrose-agar medit (X 476),


*










Sumimariea of pycnidial diameters and pycnidiospore dimensions and number of septa observed in leaf specimens collected in various locations In the United States are given in Tabips 3 and 4 for Septoria obesa and Septoria chrysenthemlla, respectively. Similar information is given in Tables 5 and 6 for pyonidiospores produced in different chrysantheum varieties after artificial inoculations with isolate No, 1 of S, obesa, or isolate No. 1 of S. chrysauthemuellag respectively.







.29


TABLE 3

DIMENSIONS AND NUDMR OF SEPTA OF PYCNIDIOSPORES AND DIAM1ERS OF PYCNIDIA OF SIPTORIA OWSA SPCIMZNS COLLECTEZD AT
VARIOUS LOCATIONS IN 1THE U7ID STAIRS




Pyoniditospore Dimensions Pyoni diospore Pycaidia, Diameters
Microns Septations Microns
Specimen aeghWidth
No. a Range Meanb' Range Range Range


1 54-106 78.0 2.8-4.5 7-13 80-156

2 46-104 75.5 2,6-4,0 7-14 74-148

3 45-100 73,2 2.8-4,5 6-13 62-156

4 44-102 72,9 2,7-4,4 6-14 60-123

5 44-100 71.4 2.8-4,4 5-14 75-132

6 50-108 74.7 2,7-4,5 6-14 68-160

7 45-98 74.2 2,6-3,9 5-13 72-149

8 45-100 7389 2,7-4,3 7-14 70-140



Overall 44-108 74,2 2,6-4.,5 5-14 60-160


a'no sample size was 34 pycnidiospores or pycnidiLa,

b analysis of variance gave no significant difference in the mean length of pycnidiospores from different specimens.







0


TABLX 4

DIMENSIONS AND NZ BR OF SEPTA OF PYCNIDIOSPOBES AND DIAMEERS
OF PYCNIDIA OF SE M RIA CIRYSANTHENELLA SPECIMENS
COLLECT AT VARIOUS LOCATIONS
IN THE UNZD STA79S



Pycnidiospore Dimensions Pycnidiospore Pycnidia Diameters
Microns Septations Microns
Specimen Leg Width
No. a Range Meanb Range Range Range


1 25-70 37.7 1,9-2.9 0-5 44-102

2 25-69 40.4 1.8-3.0 0-5 46-124

3 24-50 35.5 2*0-3.0 0-5 40-96

4 22-68 39.8 1.9-2.8 0-5 51-116



Overall 22-70 38.4 1.8-3,0 0-5 40-124


aflie sample size was 34 pycnidiospores or pycnidia,

b analysis of variance gave no significant difference in the mean length of pycnidiospores from different specimens.
















TABLE 5

CCWARISOK OF PYCNIDI0ISPOIRZ DIMENSIONS ON~ VARIETIES INOCUA79D WITH ISCLA79 NO. 1


SIX CHRYSANTHEMUM OF SEPTOMI OWSA


Chrysanthemum VarietYa Ran ength in Mitcrons as


Alaska 49-101 74,.5

Gold Ball 48-100 74.1l

Illini Regal 46-96 71.7

Illini, Warpaint 52-102 76,0

Jetfire 50-114 74.0

Mary L, Hall 47-99 72.8



Overall 46-114 73.8


'Thi rty-f our pycuidiospores were measured in each variety.

bmhere was no significant difference in mean length of spores among varieties at the 5 par cent 1eve1b


31

















TABLE 6

COI!PARISON OF PYCNIDIOSPORK DMINSItOIS ON~ SIX CHRYSMNTEMUM VARIETIES
INOCULATCD W17H ISOLATE NO. 1 OF SEPTORIA CHRYSUN1EIELIA



Chrysanthemum Vari.ty0L Length in icrons
Range MeantU


Alaska 30-68 38.4

Bonnie 29-72 40.8

Delaware 28-72 39.7

Mary L. Hall 32-74 4165

Rayonnante 28-58 37,7

Yellow Shasta 25-68 37,2


Overall 25-74 38.9


ahrty-four pycnidiospores were measured In each variety.

b Ter was no significant difference in mean length of spores among varieties at the 5 per cent lvel,










DISCUSSION,.-Cavara (32) described the pyonidiospores of

Septoria chrsanthemella as being non-septatel however# the septa in this species are usually Invisible unless the spore walls are artificially stained. Hemi and Nakamura (22) describe the pycnidiospores of Septoria obesa as being obscurely septateg and these investigators used an Iodine stain In order to observe the septa.6 The pycnidiospores of S, obesa observed by the writer were all distinctly septate if well matured. It is possible that Hmi and Nakamura observed the spores just prior to their full maturity, or there may be a difference in the distinctness of septations; in the species In different parts of the world or under different environmental conditions,

The two species of Setoi on the chrysanthemum collected in the United States are Boptoria chrysanthemella and Setr obesa.

There was no significant difference in the mean length of either Septoria obesa pycnidiospores or Soptoria chrysanthemella pycnidiospores produced on six different chrysanthemum varieties. It cannot be stated that the host variety does not affect this character, as a comparatively small number of varieties was used# and they were not selected at random from the several thousand varieties In cultivation.

No significant difference was found in the mean length of either

Sqporiobesa or.Septoria chrysanthemella pycnidiospores; collected from different locations in the United States. Geographical locations were not randomly selected. Pycnidiospore length could possibly be affected by both environmental conditions and hereditary factors.

There was a considerable difference between the dimensions and number of septa of pyonidiospores and also between the pycni dia3l










. dimensions of the two Septoria species, The mean length of the

pycuidiospores of Septori a santhemella was about one-half that of

the mean length of those of Septoria obesa, and the maximum number of

septa in the pyonidiospores of the former species was the same as the

minimum in the pyonidiLospores of the latter species, The pycnidia of

S, obesa. were usually larger than those of S, chrysantheinella and also

differed markedly in shape. A comparison of the pyenidiospores and

pyonidia of the two Septoria species is shown in Table 7 and Fig. 11.



TABLE 7

COMPARISON OF' SEPTORIA OESA AND SEPTORIA CRYSAN11EIELIA SPECIMEINS
COLALECED IN THE3 UNITED STATES: PYCNIDIOSPORES AND PYCNIDIA




Morphological. Character Septoria. chess S, chrysanthemella


Pycnidiospores:
Range in length 44-108 u 22-70 u
Mean length 74.2 u 38.4 u
Range in width 2.6-4.5 u 1.8-3.0 u
Mean width 3.2 u 2.1 u
No. of septa 5-14 0-5
Shape Tapered from base Linear, Usually
or mid-region to straight or slightly
apex. Usually curved,
curved,
Pycnidia:
Range in diameter 60-160 u 40-124 u
Mean diameter 92 u 68 u


































00. WOO L



_ _G t



0 ------- !,




0 c







36


Physiology

In 1927 Hemni and Nakamura (22) investigated the effect of temperature on the growth of Septoria chrysanthemella and Septoria obesa on solid media, The results of this investigation will be discussed later in this section, The same investigators also observed the growth of the two fungi in various solid and liquid media, but these experiments were not designed to give information on the specific nutritive requirements of the two fungi,

The effects on the growth of the fungi in cultures of different carbon sources,, nitrogen sources, pH levels, and temperature levels are reported herewith, Studies were made of pycnidiospore germination, and the habits of growth of the two fungi on potato-dextrose-agar medium were compared.

IBIH(DS AND MATERIALS.--The growth of the fungi was observed on potato-dextrose-agar medium; water was used as a medium in sporegermination studies. The culture medium employed otherwise was a modification of a basal synthetic medium described by Hacskaylo et al,

(19)e* The formula for the preparation of one liter of this medium and the compounds used unless otherwise stated are as follows:

Carbon source 10.0 g (dextrose)
N--- 425 mg (2.000 g. L-asparagine)
K112P0D4 1.0 g
M9S04.7H20 0.50 g
Fe+44 0.2 mg C1,006 mg, Pe2(S04)309H203
Zn444 0.2 mg (,8796 mg, ZnSO4.7H 20)
Mn.4 0,1 mg (,4077 mgO MnSO4.4H20)
Biotin 5 ug
Thiamine 100 ug
H20 (Distilled) to make 1 liter of solution.
Twenty grams of agar per liter of solution were used when solid
media were prepared.






37


Stock solutions of the iron# zinc, manganese,, and vitamin

sources were prepared at concentrations 1000 times those used in the culture media.

The medium described above will hereafter be referred to as synthetic liquid medium ors if agar is added, as semi-synthetic agar medium,

In all experiments In which synthetic liquid medium was used,

each replication of the medium was inoculated with the fungus by adding

5 drops of a heavy suspension of germinated pycnidiospores, and each culture was agitated once a day by shaking., The spore suspensions were prepared by flooding one-month-old potato-dextrose-agar sporulating cultures of the fungi with sterile distilled water. A different isolate was used in each replication of the experiments concerned with pH,, temperature,, carbon source,, and itrogen source. Filter papers were weighed individually after drying in an oven for 2 days at 440C, The mycelium was collected on these weighed filter papers with the aid of a Buchner funnel under vacuum, The papers and collected mycelium were weighed after drying and the differences computed. All weights of mycelium were determined to the nearest 0,1 mag.

The effect of hydrogen-ion concentration on the growth of the two fungi in synthetic liquid medium was investigated at pH units of

2 through 10. The PH1 of the medium was adjusted after autoclaving by adding measured amounts of 0.lNr lNO and 5N HCl or NaOH to 125 ml. portions of the medium in 250 ml erlemeyer flasks, The amounts of acid or base necessary to give the desired pH level were previously determined with the aid of a Beckman pH meter. Each replication was






38


inoculated with 3-day old germinating pycnidiospores and incubated for 28 days at 200C, Dry mycelial weights were then determined, Four replications were used for Se toria chrysanthemella and 6 for Setr obesa, The experiments were not conducted concurrently for the two species of Septoria, but all conditions were duplicated as nearly as possible,

The effects of different temperatures on the growth of the fungi in both synthetic liquid medium and semi-synthetic agar medium were investigated, Germinating spores in 125 ml portions of the liquid medium were incubated for 28 days at four-degree intervals from 40 to 320C,, and mycelial weights were then determined, Three replications were used for Septoria chrysanthemella and 5 for Septoria obesa. Infestation of the solid medium in petri dishes was accomplished by first inserting a sterile needle into a sporulating culture and then inserting the needle vertically into the center of the medium, These cultures were incubated for 2 days at 200C, and were then transferred to constant temperature chambers at the temperatures given above, where they remained f or 28 days. The average diameter of each culture was then estimated by making two measurements through the center of the culture, Four replications of each fungus were used in the experiments.

The effects of different carbon sources on the growth of the

fungi on semi-synthetic agar medium was observed. Carbon sources used in the experiments were mannose,, D-mannitol * maltose, galactose, sucrose, dextrose, and lactose; in addition there was a control group which lacked a carbon source, All carbon sources were used at the rate of 10 g per liter, The pH was adjusted to 5.0 after autoclaving. Four replications were used for each fungus, The mean diameters were estimated as






39


previously described after incubation of the cultures for 28 days at 200C.

The effects of several nitrogen sources on the growth of the fungi in culture were studied. Semi-synthetic agar medium less biotin and thistmine and with other nitrogen sources alternated with L-asparagine was used, The quantity of each nitrogen source used was adjusted to give a concentration of 425 mg of nitrogen per liter. The compounds and quantities used per liter are given below:

L-asparagine 2.000 g
Glycine 2.277 g
L-leucine 3,977 g
L-arginine 1.321 g
Amonium sulfate 2,014 g
Potassium nitrate 3,066 g
Control-nitrogen compounds omitted

Four replications for each fungus were used, The pff was adjusted to 5,0 after autoclaving. Incubation was for 28 days at 200C,

Germination studies were made on pycuidiospores produced in potato-dextrose-agar medium, The percentage of germination on water agar at various temperatures was determined by observing 50 spores at each temperature, Observations on pycnidiospore germination in several kinds df media were made.

RESULTS-The optimum pH for the growth of both Septoria obesa and Septoria chrysanthemella In synthetic liquid medium was pH1 5 to pH 7. No growth occurred at pH1 2 or VU1 10, and little occurred at pH1 3 or pH1 9, Summaries of the growth of S, obesa and S, chrysanthemella are given in Tables 8 and 9, respectively.






40


TABLE 8

THE EffECT OF HYDR0(EN-I(1N CONCENTRATION ON~ THIE GRCOI2 OF
SEPTORIA WEBSA IN A SYNTHETIC LIQUID MEDIUM


Dry Mycelial Weight in %,7 Mean
PH Range Mean" Final PH


2 no growth occurred

3 45,0-57,.9 53.4 3.3

4 41.6-81.7 54,6 4.5

5 98.0-169.5 129.6 6.9

6 109,8-133,7 123.9 7.1

7 73.4-125.2 99.0 7.2

8 46.0-71.1 60.2 7.7

9 31,4-40,0 36.2 8.6

10 no growth occurred


'After 28 days of growth.

b
Six renlications were used,






41


TABLE 9

THE EFFECT OF HYDR0GEN-IOIK CONENTATION ON VIE G1I(MTH OF SEPTORIA CHRYSANTHEMELLA IN A SYNTHETIC LIQUID MEDIUM


Dry ycelial Weight in UMa
PH Rage anFinal pH


2 no growth occurred

3 36,0-44,2 40.0 3.4

4 47,0-56,6 50.9 4.5

5 92.8-105.0 99.2 6.2

6 99,2-111,3 105.2 6.9

7 89,6-100,.0 95.6 7.0

8 43.0-55,4 49.0 7.7

9 26,0-35,.6 31,2 8.6

10 no growth occurred


'After 28 days of growth. bSxreplications were used.






42


The amount of growth of Septoria obesa in synthetic liquid media and on semi-synthetic agar medium at various temperatures is shown in Tables 10 and 11, respectively., The results of similar experiments with Septoria chrysanthemella are shown in Tables 12 and 13. S, obesa and S, cbrysanthemella in culture on semi-synthetic agar medium at optimum temperatures are shown in Fig, 12.


TABLE 10

THE EFFECT OF 7EMPERATUIIE ON THE GROWTH OF SEPTORIA OBESA IN SYNTHETIC LIQUID MEDIA




Dry Mycelial Weight in mg ab
OC Range Meanb


4 33,6-42,2 39.0

8 69.2-78,5 73.9

22102,0-114,2 109.5

16 - 128,4-136,4 132.3

20 118,2-131.4 125.0

24 80,2-88,5 84,9

28 25.4-30.2 27.7

32 no growth occurred



aAfter 28 days of growth.

b Five replications were used,

















113 EFFECT OF


TABLE 11

72ERMI ON THE GROM OF SPVRIA 08SA ON SM-YN7HEITIC AGO MEDIUM


Culture Diameter in mma
PC ange qa Sporulation


4 2.5-3.5 3.0 no

8 505-6,5 690 yes

12 7,0-8.0 7.4 yes

16 10.5-12.0 11.0 yes

20 12,0-14,5 13.4 yes

24 15,0-16,5 15.8 yes

28 12.2-14.4 13.2 yes

32 no growth occurred


"After 28 days of growth. bFour replications were used*


43






44


TABIZ 12

THE EFFECT OF TEMPERATURE ON 'DIR GROWTH OF SEPTORIA
CHRYSAII2HEMELLA IN SYNTHETIC LIQUID MEDIUM


Dry Mycelial Weight in g
OC Range Meanb


4 22.4-25.0 23.7

8 61.2-62.8 62.0

12 94.0-102,2 97.6



20 93.4-109.0 100.6

24 71.8-78.6 75.6

28 61.0-64,5 62.7

32 no growth occurred


aAfter 28 days of growth. b Tre replications were used,






45


TABLE 13

1THE EFFECT OF 7MPRAT~MM ON THE GROWTHI (F SEPTORIA
CHRYSANTHEBIA OII SBMI-SYNTHBTIC AGAR MEDIUM


Culture Diameters In iui5
OC Range Msb Sporulation


4 1,5-3,0 2.3 no

8 5.5-7,0 6.1 yes

12 10,5-12.0 11.3 yes

16 16.0-19.0 17,0 yes

20 23,5-28,5 26.0 yes

24 25.0-30.0 27.,6 yes

2 -31.5-35.5 33.5 yes

32 no growth occurred


&After 28 days of growth. bFour replications were used,






4G


28 '284








24 24*








209 20*







Fig. 12,--Growth of Septoria obesa (left) and Septoria chrysanthemella (right) on semi-synthetic agar medium at opt.nIrw temperatures at the and of 40 days.






47


At temperatures of 40 to 200C. in the liquid medium, both of

the fungi produced black or greenish-black, compact masses of mycelium which tended to adhere to the bottoms or sides of the vessels, At 240 and 280C* a colorless,, film-like mycelium was formed which tended to float on the surface of the liquid.

Temperature had no apparent effect on the color of cultures of either fungus grown on the solid medium.

Both Septoria obesa and Septoria cbrysanthaaella sporulated on all carbon sources used and also In the absence of carbon sources, Data an culture diameters are given in Tables 14 and 15,


TABIR 14

THE EFFECT OF CAIU3O SOURCE ON THE GRONTI OF SEPTORIA
(WSA ON SM-SYNTIIETIC AGIR ]MEDIUM



Carbon Culture Diameter in mma
Soureb RngeN Sporulation


Manno so 7.0-12.0 10.1 Yes

D-manmitol 7,0-8,5 8.0 Yes

Maltose 8,5-10.5 9,7 yes

Galactose 8,5-11,5 10.5 Yes

Sucrose 9,0-11,5 9.9 Yes

Dextrose 10.0-11.0 10.,4 yes

Lactose 5,*5-8,0 6.9 yes

Minus carbon source 3.5-7.0 6,0 yes


Afte 28 days of growth. bAt the rate of 10 g per liter. 0lour replications were used.







48


TABLE 15

THE ZFFCT OF CARBON SOURCE ONI THE GROWTH OF SEPTORIA
CHRYSANIPAEA ON( SEM1-SYNMITIC AGAR MEDIUM


Carbon Culture Diameter In mma
Source Ranuge MeanC Sporulation


Mannose 32,5-34,5 33.6 Yes

D-mannitol 25,0-27,0 26.(J Yes

Maltose 32.5-33*5 33.2 Yes

Galactose 35.0-37,0 36.1 Yes

Sucrose 35.5-43.5 40.6 Yes

Dextrose 35.5-37.0 36.2 Yes

Lactose 24,0-25,.5 24.9 Yes

Minus carbon source 16,0-18,5 17.2 Yes


aAfter 28 days of growth.
bAt the rate of 10 gper liter.
CFour replications were used*


Septoria obesa cultures were very dark or black on all nitrogen sources used and also on media lacking nitrogen; however# with glycine and also with the absence of nitrogen, the cultures were less raised and much less dense than with the other nitrogen sources., Pycaidiospores were produced in every culture but were very sparse when glycine was used and when nitrogen was lacking, The amount of growth of this fungus on the various nitrogen sources is given in Table 16.






49


TABLE 16

THE EFFECT OF NITROGEN SOURCE ON THE GROWTHI OF SEPORIA MESA ON SEMI-SYNTHETIC AGAR MEDIUM!



Nitrogen Culture Diamneter in mma
Source ~ PRaage Meanc Sporulation


Glycine 4,5-6.0 5.1 Yes

L-asparagine 11.5-13.5 124.8 Yes

t,-leucine 4.5-6,0 510 Yes

L-arginine 8.0-9.15 8.9 Yes

Ammonium sulfate 7.0-8.0 7,5 Yes

Potassium nitrate 7,,0-11,0 8,3 Yes

Minus nitrogen 5,5-6,5 6.1 Yes


aAfter 28 days of growth.
bAll nitrogen sources were
per li ter,
cFour replications were use


adjusted to give 425 mg of nitrogen


Septoria chrysanthemalla grown on artificlz~a media containing different nitrogen sources varied markedly in both size and color. The diameters of the cultures are given in Table 17, When L-asparagine was used as a nitrogen source the culture was black, compact and raised, as is typical of this fungus grown on potato-dextrose-agar. With glycine, the cultures were conspicuously concentrically zoned with narrow dark and light areas; zonation also occurred when L-arginine was used but was not as distinct as with glycine. Potassium nitrate cultures were black






to


with small# whitish areas dispersed near their centers. With L-leucine a white culture with dispersed black spots resulted, and ammonium sulfate gave a white fluffy culture with a greenish center, Cultures which lacked nitrogen had yellow interiors which were covered by a white mycelium. No pyonidiLospores were produced when ammonium sulfate was used or when nitrogen was lacking, and very few were formed when glycine was used. Cultures of the fungus growing on different nitrogen sources are shown In Fig, 13.



Z%=3I 17

THEZ FFECT OF NITROGEN SOUC ON TflE GROWTH OF SEPTORIA
CHRYSANTHEMELZA ON SEMI-SYNI'HBTIC AGAR MEDIUM!



Nitrogen Culture Diameter in mauia
Sourceb Range mean Sporulation


Glycine 14,5-18,5 15,8 Very little

L-asparagine 32,5-33.5 33e3 Yes

L-leucine 20,0-22,0 21.0 Yes

L-arginine 26,0-27,0 26,6 Yes

Ammnonium sulfate 17,0-21,5 18.19 NO

Potassium nitrate 19,5-24,0 22.0 Yes

Minus nitrogen 15,0-16,5 15.6 NO


aAf tar 28 days of growth.


b
All nitrogen sources were
per liter,
cFou replications were use


adjusted to give 425 mg of nitrogen






51


1 2 /






3 / 4






56





7




Fig. 13,--Growth characteristics of Septoria chrysanthemella on semi-synthetic agar medium supplied with different nitrogen sources (1) L-asparagine,
(2) L-arginine, (3) glycine, (4) potassium nitrate,
(5) ammonium sulfate, (6) L-leucine, (7) minus nitrogen.


4







52


Both Septoria obesa, and Septoria chrysanthemella grow slowly on potato-dextrose-agar medium. Both cultures are black, raised, roughsurfaced and compact. S, obesa usually makes more vertical growth, and S. chrysanthegnella spreads more rapidly on the surface of the medium.

The manner of germination of pycnidiospores of Septoria obesa and Septoria chrysanthemella on water agar differs from that of spores submerged in water, Onl water agar and on potato-dextrose-agar medium the spores swell, leaving constrictions at the septa and frequently between the septa, The first germination hyphae formed usually extend axially from the basal and apical cells of the spores but lateral hyphae arise a few hours later from other cells of the spores (Fig, 14). Pycnidiospores germinating in water,, potato-dextrose broth, or synthetic liquid media do not swell greatly or become conspicuously constricted, and they germinate mainly by hyphae extending axially from the basal and apical cells (Fig. 15),* Occasionally a pycnidiospore germinating in water will produce lateral hyphae during the early stages of germination, but this usually does'not occur until germination Is well advanced and the spore has become almost indistinguishable from the mycelium,

There is also a difference in the rate of germination of pycnidiospores in liquids and on solid media, Septoria chrysanthemella pycnidiospores on water agar at 200C, begin to germinate in 7 hours, and there may be 100 per cent germination at the end of 24 hours. In water, germinated pycnidiospores of this fungus are rare at the end of 24 hours and the percentage of germination is usually 50 or less at the end of














p







/



I


-f
1


Fig. 14,--Pycnidiospores o-*' cp' chrysanthemlla germinating on solid medium (X 1000).


/

/

I

d~/7P/\


//
'II


r -- I --Pycnidiospores of Septoria nieliz --erinating in water (X 1000).


chrysanthe-


53







54


48 hours* Pycnidiospores of Septoria obesa on water agar at 200C, may germinate in very low percentage after 5 hours, and the percentage of germination may be almost 100 at the end of 24 hours. One hundred per coat germination of spores of this fungus was not observed, even after 5 days on water agar, The germination rate in water is as de scribed for Septoria chrysanthemella.

The percentage of germination of pycnidiospores of the fungi on water agar at various temperatures is given in Table 18.


T-4=1 18a

PERCENTAGE OF GERMINA~TION OF PYCNIDIOSPORES ON WMER
AGAR AT VARIOUS 7EMRATUIR3S


S. dhrysantliemella S. obesa
Time in Hours Time in Hours
0 C 10 20 48 10 20 48


4 0 0 0 0 0 0

8 0 0 76 0 10 84

12 0 6 88 0 16 92

16 8 92 98 4 90 96

20 18 96 96 20 96 98

24 16 100 0 20 96 94

28 52 100 0 16 94 96

32 0 0 0 0 0 0










DI8CUSSION.-The optimum pH for the growth of both Septoria

obesa and Septoria chrysanthemella was found to be from pH 5 to pH 7. \ These findings are not exceptional, as most fungi which have been investigated grow best In slightly acid media,

Both fungi made relatively poor growth In liquid medium at 240\) and 280C, whereas maximum growth on solid medium was at these same temperatures, The poor growth at the higher temperatures in liquid medium could possibly be related to the lower retention of oxygen and other gases by liquids at higher temperatures, or chemical changes unfavorable to the fungi may have occurred at higher temperatures. Neither fungus grew at 320C, With regard to Septoria chrsanthemella, this is in conflict with the findings of Heammi and Nakaamura (22) that this fungus made better growth at 320C,, than at 200C, This difference could be due to experimental errors or to slight physiological differences between the American isolates and the Japanese isolates.

Each of the two fungi grew and sporulated on all carbon sources used and also in the absence of a carbon source. Growth of both of the species was poorest when carbon was lacking and when lactose was used as a carbon source. There was apparently very little difference in the rate of growth of either of the fungi on the several other carbon sources used.

Both Septoria obesa andSeptoria chrysanthemella grew more

rapidly when L-asparagine was used as a nitrogen source than when other nitrogen sources were used. The growth of both fungi was relatively slow on glycine, ammonium sulfate, and in the absence of nitrogen. Septoria






56


c rsanthemella was sensitive to the type of nitrogen source, e~daibiting various colors and patterns in culture and failing to produce pycnidiospores in the absence of nitrogen or with atmnonium sulfate* Whether or not the nitrogen source requirements of this fungus are related to its selective pathogenicity on chrysanthemum varieties has not been investigated,

There was little or no difference in the rate or manner of germination of the pycnidiospores of the two fungi in water or on solid media,






87


Pathogeiicity

In numerous inoculation experiments all isolates of Septoria

obesa and all isolates of Beptoria chrysanthemella produced characteristic disease symptoms on certain chrysanthemum varieties, whereas check plants remained healthy. In all cases, pycuidiLospores characteristic of the Septoria species involved were abundant in the disease lesions of the host foliage.

It has been reported by Grain and Weber (17). Raff (30), Bobinig

(3), and Fischer (16) that chrysanthemum varieties vary in susceptibility to septoria leafapot. However, all of these reports were based on observations and none were made under controlled conditions by artificial inoculations.

The pathogenicity of Septoria obesa and Septoria chrysanthemella on twenty-two varieties of chrysanthemumn were investigated in a series of inoculation experiments conducted under variable conditions in the greenhouse and in the field.

M87HODS AND MA1ERALS.-All plants in the greenhouse were grown in 4-inch, clay pots containing soil which had previously been disinfected with methyl bromide. The soil in outdoor plots was not treated; however, no chrysanthemum had been grown in the immnedi ate vicinity for several years. Fungicides were not used on experimental plants, but propagation stock was treated with a ferban spray about once a week, Malathioa or nicotine sulfate sprays were used as needed for insect control on all plants,










Plants in the greenhouse experiments were inoculated by

atomizing both surfaces of all leaves with spore suspensions made from pure cultures of the isolates. lImediately after inoculation the plants were placed in a mist chamber for 3 days and then removed to a greenhouse bench, The positions of the plants In the mist chamber and on the greenhouse bench were always randomized. A control group consisting of one plant of each variety used in the particular experiment was not inoculated with spores of these fungi but otherwise received the same treatment as did the inoculated plants.

Plants in experiments conducted in outdoor plots were inoculated with spore suspensions applied with a pressure sprayer, Control plants were covered by plastic bags during the inoculation operation,

In evaluating the severity of the disease produced by Septoria

chrysanthesnella, only the number of lesions formed was considered, Spots produced by this fungus seldom exceeded one centimeter in diameter, The scale used considered the average number of spots per leaf on the lower four leaves, Four classes were used in this rating as follows:

Severe: Average of 8 or more spots per leaf.
Moderate: Average of 3 to 7 spots per leaf,
Mild: Average of 1/4 to 2 spots per leaf.,
Healthy: No infection,

The severity of the disease caused by Septoria obesa, cannot be accurately judged by the number of lesions per leaf , as single spots frequently cover one-quarter or more of a leaf. After observing the disease produced in the first Inoculation experiment, the following scale was formulated which subsequently proved to be applicable in all other inoculation experiments with S. obesa:






59


Severe: Most of the inoculated leaves infected,
Mild: Only the lower leaves infected,
Healthy: No infection,

Other details of methods employed are given below for each experiment, The experiments are not necessarily numbered in chronological order,

Experiment 1.-Four plants each of 15 chrysanthemum varieties

propagated in the greenhouse the previous spring were removed from pots in August, 1958, and randomized in a garden plot. The plot consisted of 4 rows 3 feet apart, and plants in rows were spaced 18 inches apart. Three plants of each variety were inoculated on September 10 with isolate No. 1 of Septoria obesa. The plants were kept wet by a sprinkler for 3 days following inoculation. Disease severity was evaluated 5 weeks later.

Experiment 2.--Four plants each of 22 chrysanthemum varieties in the flowering stage were inoculated in two groups of 11 varieties each in the greenhouse on October 15 and 18, 1958, with isolate No, 1 of Septoria obesa. Disease severity was evaluated 6 weeks later,

Experiment 3.--Four young plants each of 22 chrysanthemum

varieties were inoculated in the greenhouse in two groups of 11 varieties each with isolate No. 2 of Septoria obesa on March 13 and 16, 1959. All plants were rooted cuttings with 4 to 6 expanded leaves on each plant. Disease severity was evaluated 5 weeks later,

Experiment 4.--Four plants each of 22 chrysanthemum varieties in the flowering stage were inoculated in two groups of 11 varieties each in the greenhouse on November 9 and 12, 1958, with isolate No. 1 of Septoria chrysanthemella. Disease severity was evaluated 6 weeks
later.






60


Experiment 5.--Four young plants each of 22 chrysanthemum varieties were inoculated in two groups of 11 varieties each with isolate No, 2 of Septoria chrysanthemella on March 6 and 9, 1959. All plants were rooted cuttings with 4 to 6 expanded leaves on each plant. Disease severity was evaluated 5 weeks later.

Experiment 6.--Five plants each of 14 chrysanthemum varieties were removed from pots and planted with 1-foot spacings in a garden plot early in April, 1959. (On April 15, four plants of each variety were inoculated with isolate No. 4 of Septoria chrysanthemlla, and the plants were kept wet for 3 days afterwards by means of a sprinkler. The plants were six weeks old when inoculated. The control group consisted of 1 plant each of the varieties. Disease severity was evaluated 5 weeks after inoculation.

RESULTS,*--Experiment 1,*-All chrysanthemum varieties artificially inoculated in the field with Septoria, obesa became severely diseased, Most of the control plants had a few lesions, but these were confined to leaves near the soil. The data are given in Table 19,

Experiments 2 and 3,--Young and old plants in the greenhouse were severely infected by Septori obesa, irrespective of the chrysanthemum variety. Data on these two experiments are summarized in Table 20.

Experiments 4 and 5.--Septoria chrysanthemella attacked young and old plants in the greenhouse with equal severity, but there were definite differences in the pathogenicity of this fungus on various chrysanthemum varieties, Data are shown in Table 21,

Experiment 6.--There were marked differences in disease severity on different chrysanthemum varieties artificially inoculated in the field with Septoria chrysanthemella., Data are shown in Table 22.






61


TABLE 19

SEVERITY OF? DISEASE ON MATURE PLANTS OF FIP2UMN CHRYSANTHEMUM
VARIETIES INOCUIATOD WITH SEPTORIA, CUESA IN THE FIELD




Chrysanthemum No. of No. of Plants in Each Disease Class
Variety Plants Severe Mild Healthy


Alaska 3 3

Blue Chip 3 3

Copperhead 3 3

Dark Buckley 3 3

Illini Igloo 3 3

Ill1ini Regal 3 3

Indianapolis Bronze 3 3

Jetfire 3 3

Mary L. Hall 3 3

Pink'Dot 3 3

Portrait 3 3

Rayonnante 3 3

Seneca 3 3

White Top 3 3

Yellow Shasta 3 3

Control Plants 15 10 5






62


TABLE 20

SEVRITY OF DISEASE ON T.YENTY-TATO CHRYSANTH5M VARIETIE S
INOCU1A7D WITH SEPTORIA (MESA IN THE MIENOUSE


Chrysanthemum No. of No. of Plants in Each Disease Class
Variety Plants Severe Mild Healthy


Alaska 8 8
Beauregard 8 8
Blue Chip 8 4 4
Bonnie 8 7 1
Copperhead 8 8
Dark Buckley 8 8
Delaware 8 8
Gold Ball 8 7 1
Humdinger a 7 1
Illini Igloo 8 8
Illini Regal 8 6 11
Illini Warpaint 8 7 1
Indianapolis Bronze 8 7 1
Jetfire 8 7 1
Mary LHall 8 71
Pink Dot 8 6 2
Portrait 8 8
Rayonnante 8 7 1
Seneca 8 8
White Top 8 8
Yellow Queen 8 8
Yellow Shasta 8 8
Control Plants 44 3 41






63


TABLE 21

SEVERITY OF DISEASE ON TWENTY-TWO CHIRYSANTHEMUM VARIETIES INOCULATED WITH SEPTORIA CHRYSANTIE3ELIA IN THE GREENHOUSE



Chrysanithmumi NO. Of No, of Plants in Bach Disease Class
Variety Plants Severe Moderate. R~Ld Healthy


Alaska 8 1 5 2
Beauregard 8 4 3 1
Blue Chip 8 2 4 2
Bonnie 8 7 1
Copperhead 8 5 3
Dark Buckley 8 35
Delaware 8 6 2
Gold Ball 8 3 4 1
Humdinger 8 5 3
Illini Igloo 8 4 4
Illini Regal 8 4 4
Illini Warpaint 8 2 6
Indianapolis Bronze 8 1 4 3
Jetf ire 8 3 5
Mary L. Hall 8 8
Pink Dot 8 6 2
Portrait 8 1 3 4
Rayronnante 8 6 2
Seneca 8 1 2 5
White Top 8 6 2
Yellow Queen 8 4 4
Yellow Shasta 8 1 7
Control Plants 44 44






64


TABLE 22

SMVRITV OF DISEASE ON FOURZEN CHRYSANTHEMUM VARIETIES INOCMLA78D
WITH SEPTORIA CHRYSANTHEPAELLA IN 7HE FIELD



Chrysanthemum No. of No. of Plants In Each Disease Class
Variety Plants Severe Moderate Mild Healthy


Alaska 4 2 2

Beauregard 4 2 1 1

Donnie 4 2 2

Dark Buckley 4 1 3

Delaware 4 3 1

Gold Ball 4 3 1

Illini Regal 4 2 2

Indianapolis Bronze 4 1 2 1

Jetfire 4 4

Mary LHall 4 3 1

Portrait 4 1 1 2

Rayonnante 4 2 2

White Top 4 2 1 1

Yellow Shasta 4 1 2 1

Control Plants 14 5 9






65


DISCUSSION-Setoria obesa was very pathogenic on both young and old plants of all of the 22 chrysanthemum varieties Inoculated. Whether or not any of the several thousand chrysanthemum varieties grown today possess any degree of resistance to S. obesa cannot be predicted* However,, it is reasonable to suppose that a large majority of chrysanthemum varieties is susceptible to this pathogen*

2e2toria chrysanthemella Infected young and old plants of the

same chrysanthemum variety equally, but the pathogenicity of this fungus varied greatly on different chrysanthemum varieties, The varieties Mary U, Hall and Bonnie alone were consistently, severely infected, whereas a few others were never more than mildly infected, There is apparently no doubt that the pathogenicity of S. chrysanthemella varies on different varieties of the cultivated chrysanthemum,

Septoria obesa is more pathogenic than Septoria chrsanthemella. The former species severely attacked all chrysanthemum varieties inoculated, and even a few lesions an a leaf frequently enlarged greatly, killing the entire leaf, Not only was S, chrysanthemella only mildly pathogenic on many chrysanthemum varieties,* but lesions on susceptible plants were usually less than I cm in diameter, and leaves were not killed unless the lesions were very numerous*













HOST-PARASITE~ RELATIONS

Seasonal Development of the Diseases

The development of the disease caused by Septoria obesa has been observed for the past 18 months in unprotected chrysanthemum plantings in Gainesville, Florida. In a field plot on which chrysanthemums were heavily diseased the previous fall, young shoots developed symptoms about two weeks after emerging from the soil in February and March,

Once infection has occurred, the plants remain diseased until killed by freezes the following winter, The development of symptoms and the spread of the disease are affected more by climatic conditions than by the season; however, in general,, the development of symptoms and the spread of the disease are more rapid in the spring and fall than in the summer.

The seasonal development of the disease caused by Septoria chrysanthemella was not observed, but various reports in literature indicate that its seasonal development is very similar to that of Septoria obesa.


Sources of the Inoculum

The source of primary inoculum in the field is the pycnidiospores in pycaidia imbedded in diseased leaves remaining on the ground in the fall and winter when plants are not growing, Dimock and Allen

(13) observed the disease develop late in the sumer in cut-over beds where septoria leaf spot had been controlled during the earlier part of 66






C 1


the year by spraying; they suggested that the inoculum was carried over from the previous fall*

An experiment was undertaken to determine the longevity of the inoculum on and in the soil in the Gainesville area. Field conditions were simulated as nearly as possible. The dry# lower leaves of chrysanthemum plants heavily infected with Septoria obesa were taken late in November and crumbled and mixed into field soil* This mixture was then used to form the upper inch of soil in three 5-inch pots previously two-thirds filled with field soil from another location, The pots were then moved to a location remote from chrysanthemum planting and placed in the ground with surfaces of the potted soils at ground level, The same procedure was followed with inoculum from Septoria chrysanthemella except that diseased leaves from the greenhouse were used and the operation was not begun until the middle of December., On February 4. leaf fragments were examined, and spores of both of the fungi were found by microscopic examination. On March 3. spores were still present and germinated well on water agar,

An experiment was then designed to test the pathogenicity of

the spores of each of the fungi. Three young plants of variety Jetf ire were inoculated by dipping into a water suspension of the Septoria obesa spores. Two sets of three each of control plants were used, all of variety Mary L, Hall. Onie control group was dipped into a suspension of field soil collected at a location remote from chrysanthemum plantings. The other control group was inoculated with a water suspension of S, obesa spores from culture, After inoculation all plants were maintained in a mist chamber for 3 days and then were removed to a







68


greenhouse bench, The same procedure was followed, and at the same time, with Septoria chrysanthemella except that the chrysanthemum variety used was Mary L. Hall.

All of the plants inoculated with the suspension containing the diseased leaf fragments became infected. Disease lesions were not numerous on these plants, but pycnidiospores characteristic of the species of Septoria involved were eventually produced. None of the plants dipped into the suspension of field soil became infected. All plants inoculated with spore suspensions from culture became heavily infected.

On April 5 no spores could be found in the over-wintered inoculum. However, the inoculation experiment as described above was repeated, but the only plants that became infected were those inoculated with spores from culture.

It appears to be very doubtful that inoculum in diseased leaves left on the soil in the fall remains viable into the late spring or into the summer in the Gainesville area.

After plants have become infected, pycnidia and pycaidiospores are formed in the leaf lesions. The pycnidiospores produce secondary infection when disseminated to other leaves or other plants if conditions

are f avorable.


Spore Dissemination

It has been noted by many observers that new infections of chrysanthemum plants by septoria diseases follow periods of rainy weather, According to Dimock (10) the pycnidiospores are disseminated by splashed or wind-blown water, or by various mechanical means when






69


the foliage is wet. He found that direct splashing of spores from the soil may reach a height of 16 to 18 Inches* Dimock (11) also found that the spores were dissemiinated by Insecticidal sprays and that most Insecticides commnonly used have little or no effect on the viability of the spores.


Climatic Conditions Affecting Infection and Disease Developmnent

According to Dimock (10)9 pyonidiospores must be in contact with the wet abaxial surface of a leaf for at least 24 hours before infection is established; the 24-hour wet period does not have to be continuous for infection to occur but may alternate with dry periods such as may occur with nightly dews and sunny days.

The writer agrees that some infection by each of the Septoria species may occur after a 24-hour wet period for the pycnidiLospores on the abaxial surface of the leaf, However,, several experiments In which inoculated plants were maintained in a mist chamber for only one day resulted in little or no infection. Furthermore, only an occasional germinated spore of either fungus was observed on cleared leaves which had previously been inoculated and kept wet for 24 hours at 200OC; even after 48 hours only about one-half of the spores had germinated,

Extremely hot weather retards the development of the diseases, both in the field and the greenhouse. All Inoculation experiments carried out in the greenhouse during the hot siumer months either failed completely, or else the development of symptoms was arrested until a period of mild weather arrived. In one case plants inoculated with Septoria obesa did not show disease symptoms until 6 weeks later. The






70


day temperatures in the greenhouse during sunny summer days usually ranged between 850 and 950F. During mild weather symptoms of both diseases usually become visible in 12 to 18 days and pycnidiospores are present about 1 week later. This agrees with Dimock (10) who gives the incubation period at 10 to 14 days,* with spores developing

4 to 6 days later,

It was noted by Halsted (20) in 1891 that moisture greatly favors the development of septoria leafspotso and this has been frequently observed since that time., Not only are the spores disseminated during rainy periods,, causing new infections,, but lesions already present increase in size*


Host Penetration

According to Dimock (10, 12) infection by Septoria obesa or

Septoria chrysanthemella occurs almost exclusively at the abaxial surfaces of the leaves, In several inoculation experiments carried out with plants under bell jars over water, the writer found this to be true; only rarely did infection occur when only the adaxial surfaces of leaves were inoculated. However, literature reveals no information on the specific mode of host penetration by either fungus; consequently, a study of this was undertaken,

METHODQS AND MTRIAL=.--After much experimentation with various leaf-clearing and staining techniques# the best method, and also the most rapid$ was found to be a combination of the method used by Peace

(29) to clear dead leaves and a technique described by Diener (9) for staining fungus spores and hyphae on leaves.







71


Equal volumes of crystals of chloral hydrate and phenol were placed in a beaker and heated gently until melted; a small vial was then one-half filled with a portion of this mixture,

Qie-centimeter squares were then cut fromn leaves of susceptible chrysanthemum plants previously heavily inoculated and maintained under bell jars over water for 3 to 5 days. Several squares of leaf tissue were then placed into the mixture in the vial and heated to near the simmering point until the tissue became translucent, This usually required less than 30 minutes. The leaf tissue was than soaked in warm lacto-phenol for at least 15 minutes, A square of the tissue was then placed on a glass slide, flooded with lacto-phenol deeply colored by a I per cent cotton blue solution, and heated to the simmering point for 2 or 3 minutes, The material was then examined under the microscope, and if staining was inadequate the slide was heated again. The slide was then flooded with unstained lacto-phenol * and a cover slip was placed over the material. When not being studied, the leaf material was stored in lacto-phenol.

OBSERVATIONIS AND DISCUSSION. -Numerous observations were made

in this study, both on the abaxial and adaxial surfaces of the inoculated leaves, The spores germinated in the manner and apparently at about the same rate as previously described in the section on physiology for the germination of spores in water.

Oni the abaxi al surface, germination hyphae frequently enter stomata; however, the hyphae apparently are not attracted to stomataa, as they much more frequently passed directly over them. No evidence of penetration directly through epidermal cells was observed.









Although the adaztal epiderais of chry, o tmm leaves hms s, few scattermed stomata, penetratin mw not observed. However, it Is not urso a to suppos0 that penetration through the stomata at

this epidermis occasioally occurs.

Penetration of stomata by a!2~2obes Snd ftedE! d 2 "nthamella bYPhas ame somn In Figs. 16 and 17, respectively.




NopOlgcal CharaCterS of the uycelia and pymidia of the two pathogens in the leffes of the host have Already been described In this paprw. fte study given below is concernd only with the anatomical relationships at fungous ad host tissues.

I31 IW AND NM'fIALS.-ObsewatIone are based On the study of permanent slides of lesions and adJaet tissues at host leaves. MWe leaf materia mas killed and tind for at least 48 hours in tornallnacetic-alcohol (10 parts 95 per cout ethanole 1 part glacia acetic acidg 2 parts tormain,, 7 parts water) ad embdhded in paraffin. ferial setions 10 microns thick er md id ith a rotary rAcrotome. fte sections m then fixd to glass. slides with lisupt's aftesive and eitheW left unstained or stained with safrain, satruai and fast green, Ironp'hemto~ying or' cotton blue. In pumea safrain was best tar staining the cycelimsu whereas pganidi, ow best observed unstained. All sections weoo mouted in balsam.

WIVMATIOMI ANqD DISCUWSICI.-Te invasion of halthy boat tissue by the iqoelia, of Wof one ad fttcd Is apparently idetca1. Ume uceim gows Intarellularlys nurroundng























.4


0


10 -*
Fig. 16,--Stomata. penetration by a hypha from
a germinating pycnicliospore of Septoria obesa (X 476).


/


V


N


a! '3


Fig. 17.-Stomata. penetration by a hypha from
a germinating pycnicliospore of Septoria chrysanthemella (X 476),


73


I
S






74


the host cells and causing their death, Ho evidence of direct penetration or haustoria formation was observed, The Mycelium grows most freely in the spongy and palisade mesophyll but also invades the bundle sheaths and vascular tissues. Mhe mycelium was observed to extend as much as 2 imm from the necrotic tissue of the lesion. Figure lS shows the mycelium of Septoria obesa surrounding cells of the mesophyll.

Mhe mycelium surrounding lysigenous cavities adjacent to epidermal tissues sometimes forms numerous small, spherical or elliptical-shaped cells. This is apparently an early stage in the formation of a pycnidiurn. Pycnidia were present only in necrotic tissue in this material. but have been observed in adjacent chlorotic tissues in untreated leaves. Short pycnidial beaks rupture the epidermis and extend above it or sometimes extend through stomata*

Host tissue invaded by Septoria obasa and Septoria chrysanthesnella are shown in Figs, 19 and 20, respectively,






75


"V


I


-



C




~
p



4


Fig. 18,--Cross-section of host leaf showing
intercellular invasion of mesophyll tissue by Septoria obesa mycelium (X 476).





76


Fig, 19.--Cross-section through a leaf lesion showing pycnidia of Septoria obesa (X 111),


r


Fig. 20.--Cross-section through a leaf lesion
showing pycnidia of Septoria chrysanthemella (X 111).


a


WL ft-r















SUMAY


1. Two distinct septoria diseases of the cultivated chrysanthemum
are common in the United States and in several other parts of
the world where the host is grown.

2. The valid names of the causal organisms are Septoria obesa Syd.
and Septoria chrysanthemella Sacc.

3. The disease caused by Septoria obesa appears to be more common
in the United States and is also more severe than the disease
caused by Septoria chrysanthemella.

4. Symptoms of each disease are described in detail. Septoria obesa caused black or dark brown leaf lesions which are circular to
irregular in shape and indefinite in size, Septoria chrysanthmella causes black, circular leaf lesions wih are usually one
centimeter or less in diameter.

5., The morphology of the causal organisms in culture and in host
tissues is described in detail. There is a distinct difference
in average size, shape, and number of septa of the pycuidiospores
of the two pathogens. Geographical location or host variety did
not significantly affect pycnidiospore morphology.

6. The optimum hydrogen-ion concentration for the growth of each
fungus in culture was pH 5 to pH 7; no growth occurred at pH 2
or pH 10,

7. The optimum temperatures for the growth of each pathogen in
culture on solid medium was 200 to 280C,, Little growth occurred
at 40C., and there was no growth at 320C.

8, The rate and character of growth of the pathogens in culture on
various carbon sources and nitrogen sources is described,

9. Pycnidiospores germinated rapidly on solid media and germinated
slowly in water. The rate of germination at various temperatures
is described.

10. Septoria obesa severely attacked all chrysanthemum varieties
inoculated, but the pathogenicity of Septoria chrysanthemella
varied greatly on different varieties of the host,


77











11, In the Gainesville, Florida, area symptoms of the disease may
be present on chrysanthemum leaves which are two or more weeks
old during any season of the year,

12. Inoculum in diseased leaves left on and in the soil in December
did not remain viable into April of the following spring,

13, Pycnicliospores of the causal organisms are disseminated principally by spattering water. Infection progresses from the lower
leaves upward to the upper leaves of the plants.

14. Infection and disease development are favored by moisture and
cool or mild temperatures and are restricted or retarded by very
hot, dry weather.

15, Penetration of the host is by hyphae of germinated spores entering stomata of the leaves.

16, Pathological anatomy is described in detail. Mhe mycelium of
each of the causal organisms grows intercellularly and does not
produce haustoria.














LI79ERATUEE CI72D


1. Beach, B, S. 1893. Leaf-spot of chrysanthemums, Eleventh
Ann. Rept. N. Y, Agr. Exp. Sta. (Geneva). p. 557.

2. Bensancie,, M, 1927. Inventarlo das molestias das plantas
agricolas de S. Miguel. (Abs.) Rev. Appi. Mycol. 6:466.

3. Bobmig, F. 1937. TUber die Anfalligkeit der ChrysanthemumSorten Gartenwelt 41:510,

4. Brien, R, M. and Joan M, Dingley. 1953. First supplement to
11a revised list of plant diseases recorded in New Zealand."t
(Abs.) Rev. Appl. Mycol. 33:53.

5. Chiff lot, J. 1904. Maladies et Parasites du Chrysantheme.
Librairie et Imprimerie Horticoles. Paris., 56 pp.

6. Cooke, M, C, 1887. New British fungi. Grevillea 15:109.

7. Dvds, 1, 1951. Rannsoknir a grooursjukdomum. (Abs.)
Rev. Appl, Mycol. 30:559.

8. De Carvaiho, T, 1948, Relacao preliminar de doencas encontradas
em plantes e insectos corn anatacoes fitopatologicas. (Abs.)
Rev. Appl. Mycol. 29:89.

9. Diener, U. L6 1955. Host-penetration and pathological histology
in gray leaf spot of tomato. Phytopathology 45:654-658.

10. Dirnock, A. W. 1942. Controlling septoria leaf spot of the
chrysanthemum. Bull. Chrysanth. Soc. Amer, 10:6-11.

11. Dimock, A. WV. 1951. The dispersal of viable fungus spores by
insecticides. Phytopathology 41: 152-156.

12. Dimock, A, W, 1953. Control of three ills of chrysanthemum.
Plant Diseases Yearbook of Agriculture. U. S, Dept. Agree
Washington. pp. 592-595.

13, Dimock, A. W, and H. Allyn, 1944. Dipping rooted chrysanthemum
cuttings in fermate for septoria leafspot control. Bull, Chrysanth.
Soc. Amer. 12:9-11,

14. Eastham, J, W, 1937. Report of Provincial Plant Pathologist,
(Abs.) Rev. Appl, Mycol. 16:797.


70






.80


15, Ellis, J, B. and J. Dearness. 1893. New species of Canadian
fungi. The Canadian Record of Science. p. 271.

16. Fischer, R, 1938. Beobachtungen uber die Anfalligkeit der
Chrysanthemum-Sorten. Gartenwelt 42:8a.

17. Gram, E. and Anna Weber* 1952. Plant diseases in orchard,,
nursery and garden crops. Macdonald & Coo London. pp., 431-436.

18. Greene, H, C, 1949. Notes on Wisconsin parasitic fungi. Amer,
Midi. Naturalist 41:738-739.

19. Hacskaylo, J, et al, 1954, Growth of fungi on three sources of
nitrogen. MycologiZa 46:691-701.

20. Hosted, B. D. 1891. A chrysanthemum blight, Garden and Forest
4:560.

21. Halsted, B, D, 1892. Fungous troubles in the cutting beds,
Garden and Forest 5:91-92.

22. Hemmi, T, and H. Nakamura. 1927. Studies on septorioses of
plants, Memoirs of the College of Agriculture, Kyoto Imperial
Univo no. 3. 24 pp.

23. Ikata, S, 1928. Fungous diseases of the insect-powder plant.
(Abs.) Rev. Appl, Mycol. 8:174-175.

24. Loos, C. A. 1941. Some diseases of garden plants. Trop, Agr.
(Ceylon) 96:22-27.

25. Magnus,, P. 1907. Uber die Benennung der Septoria auf chrysanthemum
indicum und deren Auftreten im mittleren Europa. Berichte der
Deutschen Botanischen Gesellschaft 25: 299-301.

26, Marchal, E, 1933. Observations et recherches effectuees a la
Station de Phytopathologie do 1'Etat pendant 1'anneee (Abs.)
Rev, Appi. Mycol. 12:677.

27, Mata Quesada, B, 1950. Principles enfermedades y plagas en los
\tultivos mas comes do la Meseta Central. (Abs.) Rev, Appi.
Mycol. 30:217,

28. Niklova, H. 1949. Tni ctvrti roku zahradnicke praxe ocima
fytopathologa. (Abs.) Rev. Appi. Mycol. 29:619,

29. Peace, L. M,. 1910. Notes upon the clearing and staining of leaves and stems. Plant World 13:93-96.

30. Raff, U. 1926. Die pilzlichen und tierischen Feinde des chrysanthemum. Gartenwelt 30: 782-784.











31. Saccardo, P. A., 1884. Sylloge fungorum 3:549. 32. Saccardo3, P. A, 1895. Syllogo fungorum 11:542. 33. Saccardo, P. A, 1898. Sylloge fungorum 14:973. 34. Saccardo, P. A, 1913, Sylloge fungorzm 22:1104, 35. Salmon, E, S, and W, U. Ware. 1937. Department of mycology.
(Abs.) Rev. Appi. Mycol. 16:366.

36. Sydow, H. and P, Sydow, 1914. Zweiter Boitrag zur Kenntnis der'
parasitischen Pilzflora des nordlich Japans, Annales Mycologici
12:164.

37. Trent, J. A. 1939., The status of Cyl1bndrosporium chrysanthemi
8. & D, as the causative agent of chrysanthemum leaf blight,
Trans, Kans, Mcad. Sci. 42:203-204.

38. Voglino, P. 1901, Sopra una nalattia dei crisanteni coltivati.
Malpighia 15:329.

39. Weiss, F. 1950. Index to plant diseases in the United States,
U. S. Dep. Agr. Plant Disease Survey Special Pub, 1, part 1.
p. 169.















BIOGRAPHICAL SKETCH


Henry Thomas Waddell was born in Wilson, Arkansas, on April 19, 1918. He attended public schools in Arkansas and Tennessee and graduated from Troy High School, Troy,, Tennessee, in 1937. He served for six years with the 30th Infantry D~ivision during World War II and was separated from service in 1946 with the rank of 1st Lieutenant.

Mr. Waddell completed two years of study at the University of Tennessee Junior College, Martin, Tennessee, and continued his studies at George Peabody College for Teachers, Nashville, Tennessee, where he received the Bachelor of Science degree in 1949 and the Master of Arts degree in 1951,

He was employed as Assistant Professor of Biology by the University of Tennessee Martin Branch, Martin, Tennessee, from September, 1949, to June, 1956.

Mr. Waddell entered the University of Florida in June,, 1956,

to pursue a course of study leading to the Doctor of Philosophy degree with a major in Plant Pathology.

Hie is a member of the American Phytopathological Society,* the American Institute of Biological Sciences, Gamma Sigma Delta, and Phi Kappa Phi.


82















This dissertation was prepared under the direction of the chairman of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy. August 8, 1959




gCit4 Dean, College of Agricult-ure




Dean, Graduate School

SUPERVISORY COMMITMIE:



Chaiikan



A7




Full Text
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES iv
LIST OF FIGURES vi
INTRODUCTION 1
THE DISEASES 2
Host Range ....... ...... 2
Geographical Distribution ... 3
Economic Importance .... 4
Symptoms ..... . 5
THE CAUSAL ORGANISMS 12
Taxonomy 12
Morphology 16
Physiology ....... 36
Pathogenicity ..... 57
HOST-PARASITE RELATIONS 66
Seasonal Development of the Disease 66
Sources of the Inoculum 66
Spore Dissemination 68
Climatic Conditions Affecting Infection and Disease
Development .. 69
Host Penetration 70
Pathological Anatomy ...... 72
SUMMARY 77
LITERATURE CITED 79
BIOGRAPHICAL SKETCH 82
iii


Fig. 20.--Cross-section through a leaf lesion
showing pycnidia of Septoria chrysanthemel1a (X 111)


26
Pig. 9r-Pycnidiospores of Septoria chrysanthe-
aella (X 476).


13
be referred to under the name of Septoria chrysanthemella Sacc.t as
the name had been changed by Saccardo and not by Cavara.
In 1897 Rostrup (33) in Denmark, described a fungus parasitic
on the chrysanthemum under the name of Septoria chrysanthemi Rostr.
Due to the presence of Alleschers species, Saccardo and Sydow (33)
changed the name to Septoria rostrupii Sacc. and Syd. in 1899. Magnus
(25) and Hemmi and Nakamura (22) state that this fungus is the same
as Septoria chrysanthemella Sacc. After studying the descriptions of
the two fungi, this writer agrees with them.
In 1907 in Bohemia, Bubak and Kabat (34) described a fungus
attacking the chrysanthemum under the name of Septoria chrysanthemi-
indici Bub. & Kab. Magnus (25) believed this fungus to be synonymous
with Septoria chrysanthemella Sacc. If the original description is
accurate the opinion of Magnus is incorrect, for the pycnidiospores
of S. chrysanthemella are filiform and 0-5 septate, whereas Bubak and
Kabat described the pycnidiospores of their fungus as being tapered
from the base to the apex and 10-15 septate. According to this descrip
tion the pycnidiospores of the latter fungus are similar to those of
Septoria obesa Syd. When the type specimen of Bubak and Kabat was
examined by Hemmi (22), he found that the pycnidiospores were not as
described, being more similar to those of S. chrysanthemella Sacc.
Since more than one species of Septoria may occur on the same plant,
the synonymy of S. chrysanthemi-indicl with either S. chrysanthemella
Sacc. or S. obesa Syd. is questionable.
According to Chifflot (5) Septoria variaos Joffrin is a synonym
of Septoria chrysanthemella. The writer has not had an opportunity


36
Physiology
In 1927 Herami and Nakamura (22) investigated the effect of
temperature cm the growth of Septoria chrysanthemella and Septoria
obesa on solid media. The results of this investigation will be dis
cussed later in this section. The same investigators also observed the
growth of the two fungi in various solid and liquid media, but these
experiments were not designed to give information on the specific
nutritive requirements of the two fungi.
The effects on the growth of the fungi in cultures of different
carbon sources, nitrogen sources, pH levels, and temperature levels are
reported herewith. Studies were made of pycnldiospore germination, and
the habits of growth of the two fungi on potato-dextrose-agar medium
were compared.
METHODS AND MATERIALS. The growth of the fungi was observed on
potato-dextrose-agar medium; water was used as a medium in spore-
germination studies. The culture medium employed otherwise was a modi
fication of a basal synthetic medium described by Hacskaylo et al.
(19). The formula for the preparation of one liter of this medium and
the compounds used unless otherwise stated are as follows:
Carbon source
N
kh2po4
MgS047H20
Fe444
Zn444
Mn44
10.0 g (dextrose)
425 mg (2.000 g, L-asparagine)
1.0 g
0.50 g
0.2 mg Cl.006 mg, Fe2 (804)3*9^0}
0.2 mg (.8796 mg, ZnS047H20)
0.1 mg (.4077 mg, ¡OaSO^^O)
Biotin 5 ug
Thiamine 100 ug
H20 (Distilled) to make 1 liter of solution.
Twenty grams of agar per liter of solution were used when solid
media were prepared.


BIOGRAPHICAL SKETCH
Henry Thomas Waddell was born in Wilson, Arkansas, on
April 19, 1918. He attended public schools in Arkansas and
Tennessee and graduated from Troy High School, Troy, Tennessee,
in 1937. He served for six years with the 30th Infantry Bivision
during World War II and was separated from service in 1946 with
the rank of 1st Lieutenant.
Mr. Waddell completed two years of study at the University
of Tennessee Junior College, Martin, Tennessee, and continued his
studies at George Peabody College for Teachers, Nashville, Tennessee,
where he received the Bachelor of Science degree in 1949 and the
Master of Arts degree in 1951.
He was employed as Assistant Professor of Biology by the
University of Tennessee Martin Branch, Martin, Tennessee, from
September, 1949, to June, 1956.
Mr. Waddell entered the University of Florida in June, 1956,
to pursue a course of study leading to the Doctor of Philosophy degree
with a major in Plant Pathology.
He is a member of the American Phytopathological Society, the
American Institute of Biological Sciences, Gamma Sigma Delta, and
Plii Kappa Phi.
82


3
Fig. 1,Lesions on old chrysanthemum leaves
6 weeks after inoculation with spores of Septoria
obesa (X 1/3).


20
RESULTS.The following descriptions of the morphological
characters of the two species of Septoria under consideration are
based on the studies described above.
Septoria obesa.Pycnidia formed in host tissues are typically
globose or subglobose but sometimes pyriform or irregular in shape,
60-160 microns in diameter. Short to rather elongate beaks are present,
and the diameters of ostioles vary from almost no opening to that
measuring one-half the diameter of the pycnidium. A pycnidium of S.
obesa is shown in the leaf tissues of the chrysanthemum in Fig. 5.
Pycnidial walls are composed of 3 to 7 layers of cells which are
isodiametric to elongate in shape. The inner layer of cells is hyaline,
and the outer layers are brown. Conidiophores are composed of cells
of the inner wall of the pycnidium which have became slightly elongated;
they are hyaline, 3-7 microns in length. The mycelium is mostly 3
microns or less but sometimes up to 6 microns in diameter, finely
granular, hyaline, constricted at the septa, obtusely branched, and
septate at the points of branching. A mature pycnidiospore of S. obesa
is relatively large and rounded at the base and tapers from the basal
end or from near the mid-region of the spore to the apex (Fig. 6). In
culture on potato-dextrose-agar medium the morphology of pycnidiospores
is similar to that described above. Pycnidia vary greatly in shape,
often forming extensive flattened cavities, and the ostioles are not
well defined (Fig. 7). Most of the mycelium is devoted to producing
spherical or elliptical-shaped cells which go into the formation of
pycnidial walls


LITERATURE CITED
1. Beach, B. S, 1893. Leaf-spot of chrysanthemums. Eleventh
Ann. Rept. N. Y. Agr. Exp. Sta. (Geneva), p. 557.
2. Bensande, M. 1927. Inventario das molestias das plantas
agricolas de S. Miguel. (Abs.) Rev. Appl. Mycol. 6:466.
3. Bohmig, F. 1937. Uber die Anfalligkeit der Chrysanthemum-
Sorten Gartenwelt 41:510.
4. Brien, R. M. and Joan M. Dingley. 1953. First supplement to
"a revised list of plant diseases recorded in New Zealand."
(Abs.) Rev. Appl. Mycol. 33:53.
5. Chifflot, J. 1904. Maladies et Parasites du Chrysantheme.
Librairie et Imprimerie Horticoles. Paris. 56 pp.
6. Cooke, M. C. 1887. New British fungi. Grevillea 15:109.
7. Davidson, I. 1951. Rannsoknir a grooursjukdomum. (Abs.)
Rev. Appl. Mycol. 30:559.
8. De Carvalho, T. 1948. Relacao preliminar de doencas encontradas
em plantes e insectos com anatacoes fitopatologicas. (Abs.)
Rev. Appl. Mycol. 29:89.
9. Diener, U. L. 1955. Host-penetration and pathological histology
in gray leaf spot of tomato. Phytopathology 45:654-658.
10. Dimock, A. W. 1942. Controlling septoria leafspot of the
chrysanthemum. Bull. Chrysanth. Soc. Amer. 10:6-11.
11. Dimock, A. W. 1951. The dispersal of viable fungus spores by
insecticides. Phytopathology 41:152-156.
12. Dimock, A. W. 1953. Control of three ills of chrysanthemum.
Plant Diseases Yearbook of Agriculture. U. S. Dept. Agr.
Washington, pp. 592-595.
13. Dimock, A. W. and H. Allyn. 1944. Dipping rooted chrysanthemum
cuttings in fermate for septoria leafspot control. Bull. Chrysanth.
Soc. Amer. 12:9-11.
14. Eastham, J. W. 1937. Report of Provincial Plant Pathologist.
(Abs.) Rev. Appl. Mycol. 16:797.
79


59
Severe: Most of the inoculated leaves infected.
Mild: Only the lower leaves infected.
Healthy: No infection.
Other details of methods employed are given below for each
experiment. The experiments are not necessarily numbered in chrono
logical order.
Experiment 1.Four plants each of 15 chrysanthemum varieties
propagated in the greenhouse the previous spring were removed from pots
in August, 1958, and randomized in a garden plot. The plot consisted
of 4 rows 3 feet apart, and plants in rows were spaced 18 inches apart.
Three plants of each variety were inoculated on September 10 with
isolate No. 1 of Septoria obesa. The plants were kept wet by a
sprinkler for 3 days following inoculation. Disease severity was
evaluated 5 weeks later.
Experiment 2.Four plants each of 22 chrysanthemum varieties
in the flowering stage were inoculated in two groups of 11 varieties
each in the greenhouse on October 15 and 18, 1958, with isolate No. 1
of Septoria obesa. Disease severity was evaluated 6 weeks later.
Experiment 3.Four young plants each of 22 chrysanthemum
varieties were inoculated in the greenhouse in two groups of 11 varieties
each with isolate No. 2 of Septoria obesa on March 13 and 16, 1959. All
plants were rooted cuttings with 4 to 6 expanded leaves on each plant.
Disease severity was evaluated 5 weeks later.
Experiment 4.Four plants each of 22 chrysanthemum varieties
in the flowering stage were inoculated in two groups of 11 varieties
each in the greenhouse on November 9 and 12, 1958, with isolate No. 1
of Septoria chrysanthemella. Disease severity was evaluated 6 weeks
later


75
Fig. 18.-Cross-section of host leaf showing
intercellular invasion of mesophyll tissue by Septoria
obesa mycelium (X 476).


71
Equal volumes o crystals of chloral hydrate and phenol were
placed in a beaker and heated gently until melted; a snail vial was
then one-half filled with a portion of this mixture.
One-centimeter squares were then cut from leaves of susceptible
chrysanthemum plants previously heavily inoculated and maintained under
bell jars over water for 3 to 5 days. Several squares of leaf tissue
were then placed into the mixture in the vial and heated to near the
simmering point until the tissue became translucent. This usually
required les3 than 30 minutes. The leaf tissue was then soaked in warm
lacto-phenol for at least 15 minutes. A square of the tissue was then
placed on a glass slide, flooded with 1acto-phenol deeply colored by
a 1 per cent cotton blue solution, and heated to the simmering point
for 2 or 3 minutes. The material was then examined under the micro
scope, and if staining was inadequate the slide was heated again. The
slide was then flooded with unstained lacto-phenol, and a cover slip
was placed over the material. When not being studied, the leaf material
was stored in lacto-phenol.
OBSERVATIONS AND DISCUSSION.Numerous observations were made
in tills study, both on the abaxial and adaxial surfaces of the inoculated
leaves. The spores germinated in the manner and apparently at about
the same rate as previously described in the section on physiology for
the germination of spores in water.
On the abaxial surface, germination hyphao frequently enter
stomata; however, the hyphae apparently are not attracted to stomata,
as they much more frequently passed directly over them. No evidence
of penetration directly through epidermal cells was observed.


37
Stock solutions of the iron zinc manganese and vitamin
sources were prepared at concentrations 1000 times those used in the
culture media.
The medium described above will hereafter be referred to as
synthetic liquid medium or if agar is added as semi-synthetic agar
medium.
In all experiments in which synthetic liquid medium was used
each replication of the medium was inoculated with the fungus by adding
5 drops of a heavy suspension of germinated pycnidiospores and each
culture was agitated once a day by shaking. The spore suspensions
were prepared by flooding one-month-old potato-dextrose-agar sporu-
lating cultures of the fungi with sterile distilled water. A dif
ferent isolate was used in each replication of the experiments con
cerned with pH, temperature, carbon source, and nitrogen source.
Filter papers were weighed individually after drying in an oven for
2 days at 44C. The mycelium was collected on these weighed filter
papers with the aid of a Buchner funnel under vacuum. The papers and
collected mycelium were weighed after drying and the differences com
puted. All weights of mycelium were determined to the nearest 0.1 mg.
The effect of hydrogen-ion concentration on the growth of the
two fungi in synthetic liquid medium was investigated at pH units of
2 through 10. The pH of the medium was adjusted after autoclaving by
adding measured amounts of 0.1N, IN, and 5N HC1 or NaOH to 125 ml
portions of the medium in 250 ml erlenmeyer flasks. The amounts of
acid or base necessary to give the desired pH level were previously
determined with the aid of a Beckman pH meter. Each replication was


36
chrysanthemel1a was sensitive to the type of nitrogen source, exhibit
ing various colors and patterns in culture and failing to produce
pycnidiospores in the absence of nitrogen or with ammonium sulfate.
Whether or not the nitrogen source requirements of this fungus are
related to its selective pathogenicity on chrysanthemum varieties has
not been investigated,
Hiere was little or no difference in the rate or manner of
germination of the pycnidiospores of the two fungi in water or on
solid media.


00
15. Ellis, J. B. and J. Dearness. 1893. New species of Canadian
fungi. The Canadian Record of Science, p. 271.
16. Fischer, R. 1938. Beobachtungen uber die Anfalligkeit der
Chrysanthemum-Sorten. Gartenwelt 42:83.
17. Gram, E. and Anna Weber. 1952. Plant diseases in orchard,
nursery and garden crops. Macdonald & Co. London, pp. 431-436.
18. Greene, H. C. 1949. Notes on Wisconsin parasitic fungi. Amer.
Midi. Naturalist 41:738-739.
19. Haeskaylo, J. ot al. 1954. Growth of fungi on three sources of
nitrogen. Mycologia 46:691-701.
20. Ilalsted, B. D. 1391. A chrysanthemum blight. Garden and Forest
4:560.
21. Ilalsted, B. D. 1892. Fungous troubles in the cutting beds.
Garden and Forest 5:91-92.
22. Hemmi, T. and H. Nakamura. 1927. Studies on septorioses of
plants. Memoirs of the College of Agriculture, Kyoto Imperial
tfeiiv. no. 3, 24 pp.
23. Ikata, S. 1928. Fungous diseases of the insect-powder plant.
(Abs.) Rev. Appl. Mycol. 8:174-175.
24. Loos, C. A. 1941. Some diseases of garden plants. Trop. Agr.
(Ceylon) 96:22-27.
25. Magnus, P. 1907. Uber die Banennung der Septoria auf chrysanthemum
indicum und deren Auftreten im mittleren Europa. Berichte der
Deutschen Botanischen Gesellschaft 25:299-301.
26. Marchal, E. 1933. Observations et recherches effectuees a la
Station de Phytopathologie de l'Etat pendant l'annee. (Abs.)
Rev. Appl. Mycol. 12:677.
27. Mata Quesada, E. 1950. Principles enfermedades y plagas en los
'tuitivos mas comunes de la Meseta Central. (Abs.) Rev. Appl.
Mycol. 30:217.
28. Niklova, H. 1949. Tri ctvrti roku zahradnicke praxe ocima
fytopathologa. (Abs.) Rev. Appl. Mycol. 29:619.
29. Peace, L. M. 1910. Notes upon the clearing and staining of
leaves and stems. Plant World 13:93-96.
30. Raff, E. 1926. Die pilzlichen und tierischen Feinde des chrysan
themum. Gartenwelt 30:782-784.


34
dimensions o the two Septoria species. The mean length of the
pycnidiospores of Septoria chrysaathemella was about one-half that of
the mean length of those of Septoria obesa, and the maximum number of
septa in the pycnidiospores of the former species was the same as the
minimum in the pycnidiospores of the latter species. The pycnidia of
S. obesa were usually larger than those of S. chrysanthemella and also
differed markedly in shape. A comparison of the pycnidiospores and
pycnidia of the two Septoria species is shown in Table 7.and Fig. 11.
TABLE 7
COMPARISON OF SEPTORIA OBESA AND SEPTORIA CHRYSANTHEMELLA SPECIMENS
COLLECTED IN THE UNITED STATES: PYCNIDIOSPORES AND PYCNIDIA
Morphological Character
Septoria obesa
S. chrysanthemella
Pycnidiospores:
Range in length
44-108 u
22-70 u
Mean length
74.2 u
38.4 u
Range in width
2.6-4.5 u
1.8-3.0 u
Mean width
3.2 u
2.1 u
No. of septa
5-14
0-5
Shape
Tapered from base
or mid-region to
apex. Usually
curved.
Linear. Usually
straight or slightly
curved.
Pycnidia:
Range in diameter
60-160 u
40-124 u
Mean diameter
92 u
68 u


es
DISCUSSION.Septoria obesa was very pathogenic on both young
and old plants of all of the 22 chrysanthemum varieties inoculated.
Whether or not any of the several thousand chrysanthemum varieties grown
today possess any degree of resistance to S. obesa cannot be predicted.
However, it is reasonable to suppose that a large majority of chrysan
themum varieties is susceptible to this pathogen.
Septoria chrysanthemella infected young and old plants of the
same chrysanthemum variety equally, but the pathogenicity of this fungus
varied greatly on different chrysanthemum varieties. The varieties
Mary L. Hall and Bonnie alone were consistently, severely infected,
whereas a few others were never more than mildly infected. There is
apparently no doubt that the pathogenicity of S. chrysanthemella varies
on different varieties of the cultivated chrysanthemum.
Septoria obesa is more pathogenic than Septoria chrysanthemella.
The former species severely attacked all chrysanthemum varieties in
oculated, and even a few lesions an a leaf frequently enlarged greatly,
killing the entire leaf. Not only was S. chrysanthemella only mildly
pathogenic on many chrysanthemum varieties, but lesions on susceptible
plants were usually less than 1 cm in diameter, and leaves were not
killed unless the lesions were very numerous.


43 hours* Pycnidiospores of Soptoria obesa on water agar at 20C*
54
may germinate in very low percentage after 5 hours, and the percentage
of germination may bo almost 100 at the end of 24 hours* One hundred
per cent germination of spores of this fungus was not observed, even
after 5 days on water agar. The germination rate in water is as de
scribed for Septoria chrysanthemella.
The percentage of germination of pycnidiospores of the fungi on
water agar at various temperatures is given in Table 13.
tabie IS
HERCENTA OF GERMINATION OF PYCNIDIOSPORES ON WATER
AGAR AT VARIOUS TEMPERATURES
c
S. dhry s anthemel1a
Time in Hours
S* obesa
Time in Hours
10
20
48
10
20
48
4
0
0
0
0
0
0
3
0
0
76
0
10
84
12
0
6
88
0
16
92
16
8
92
98
4
90
96
20
18
96
96
20
96
98
24
16
100
0
20
96
94
23
52
100
0
16
94
96
32
0
0
0
0
0
0


LIST OF FIGURES
Figure Page
1.Lesions on Old Chrysanthemum Leaves 6 Weeks after
Inoculation with Spores of Septoria obesa .. 8
2.Lesions on Young Chrysanthemum Leaves 6 Weeks after
Inoculation with Spores of Septoria obesa 9
3.Lesions on Old Chrysanthemum Leaves 6 Weeks after
Inoculation with Spores of Septoria chrysanthemella ... 10
4,Lesions on Young Chrysanthemum Leaves 8 Weeks after
Inoculation with Spores of Septoria chrysanthemella . 11
5. Longitudinal Section of a Pycnidium of Septoria obesa
in Chrysanthemum Leaf Tissues ...... 21
6. Pycnidiospores of Septoria obesa 22
7. Pycnidia of Septoria obesa on Potato-Dextrose-Agar
Medium . . . . 23
8. Longitudinal Section of a Pycnidium of Septoria chrysan-
themella in Chrysanthemum Leaf Tissues ......... 25
9. Pycnidiospores of Septoria chrysanthemella ....... 26
10. Pycnidia of Septoria chrysanthemella on Potato-Dextrose-
Agar Medium ........... 27
11. Camera Lucida Drawing of Pycnidiospores of Septoria
chrysanthemella and Septoria obesa ........... 35
12. Growth of Septoria obesa and Septoria chrysanthemella
on Semi-synthetic Agar Medium at Optimum Temperatures
at the End of 40 Days 46
13. Growth Characteristics of Septoria chrysanthemella on
Semi-synthetic Agar Medium Supplied with Different Nitrogen
Sources (1) L-asparagine, (2) L-arginine, (3) glycine (4)
potassium nitrate, (5) ammonium sulfate, (6) L-leucine,
(7) minus nitrogen ............. 51
vi


c?
tli year by spraying; they suggested that the inoculum was carried
over from the previous fall.
An experiment was undertaken to determine the longevity of
the inoculum on and in the soil in the Gainesville area. Field con
ditions wore simulated as nearly as possible. The dry, lower leaves
of chrysanthemum plants heavily infected with Septorla obesa were
talcen late in November and crumbled and mixed into field soil. This
mixture was then used to form the upper inch of soil in three 5-inch
pots previously two-thirds filled with field soil from another location.
The pots were then moved to a location remote from chrysanthemum plant
ing and placed in the ground with surfaces of the potted soils at
ground level. The same procedure was followed with inoculum from
Septorla chrysanthemella except that diseased leaves from the green
house were used and the operation was not begun until the middle of
December, On February 4, leaf fragments were examined, and spores of
both of the fungi were found by microscopic examination. On March 3,
spores were still present and germinated well on water agar.
An experiment was then designed to test the pathogenicity of
the spores of each of the fungi. Three young plants of variety Jetfire
were inoculated by dipping into a water suspension of the Septorla
obesa spores. Two sets of three each of control plants were used, all
of variety Mary L. Hall, One control group was dipped into a suspension
of field soil collected at a location remote from chrysanthemum plant
ings, The other control group was inoculated with a water suspension
of S. obesa spores from culture. After inoculation all plants were
maintained in a mist chamber for 3 days and then were removed to a


'7


18
Septoria chrysanthomella
Specimen and
Isolation No.
Geographical Source
1
2
3
4
Troy, Tennessee
Beauiaont, Stexas
Ames, Iowa*
Ames, Iowa*
Specimens from Ames came from different nurseries.
Studies were made of the morphological characters of the fungi
in the original leaf samples, in pure culture, and in the leaves of
several chrysanthemum varieties artificially inoculated with pure
cultures of the pathogens.
METHODS AND MATERIALS.In the observations of pycnidiospores
in the original leaf specimens, randomization of the spores was obtained
by soaking several lesions of each specimen in water for three hours and
then stirring the resulting spore suspensions vigorously. In order to
stain the septa of the pycnidiospores, an iodine solution, was added to
the suspensions. Drops of the suspensions were then placed on glass
slides and pycnidiospores measured by use of a previously calibrated
ocular micrometer. Septoria obesa pycnidiospores were measured for
width near their bases, whereas those of Septoria chrysaathemella were
measured for width midway between their bases and apices. Lengths and
widths were measured at 440 and 970 magnifications, respectively. A
mechanical stage was used to insure that no spore was measured more
than once.
The procedure used in securing isolations from the original
leaf specimens was as follows: Hie diseased leaves were soaked in


In 1893 Ellis and Dearness (15) in Canada described a fungus
parasitic on chrysanthemums and named it Cyllndrosporlua chrysantheml
15
E. ft D. The spores of this fungus were very similar to those of
Septoria obesa Syd. and the spore-bearing structures were similar in
shape and size to pycnidia of S. obesa Sydbut lacked a pycnidial vail.
The spore-bearing organ vas therefore considered to be an acervulus
and not a pycnidlum. Trent (37) suggests that this fungus is synonymous
with Septoria chrysanthemella Sacc., and Greene (18) states that it may
be the same as Septoria obesa Syd. Unless the original description can be
shown to be wrong, the writer cannot consider this fungus to be classi
fied in the genus Septoria.
Weiss (39) lists Septoria leucanthemi Sacc. ft Spec., Septoria
macrosporia Dearn., and Septoria cercosporoides Trail as probable
synonyms of Septoria obesa Sacc. The writer cannot agree that any one
?
Of these three fungi is synonymous with S. obesa Sacc., as all three
were described on Chrysanthemum leucanthemum and cross-inoculation
experiments have not been conducted. Furthermore, the pycnidiospores
of both Septoria leucanthemum (31) and Septoria macrosporia (18) are
filiform. This differs from the pycnidiospores of Septoria obesa which
are obclavate. The pycnidiospores of Septoria cercosporoides as given
by Cooke (6) after Trail appear to be very similar to those of S. obesa
but are somewhat smaller.
The valid name of the fungus under consideration is:
Septoria obesa Syd. 1914.
Synonyms: none.


7S
11. In the Gainesville, Florida, area symptoms of the disease may
be present on chrysanthemum leaves which are two or more weeks
old during any season of the year.
12. Inoculum in diseased leaves left on and in the soil in December
did not remain viable into April of the following spring.
13. Pycnidiospores of the causal organisms are disseminated princi
pally by spattering water. Infection progresses from the lower
leaves upward to the upper leaves of the plants.
14. Infection and disease development are favored by moisture and
cool or mild temperatures and are restricted or retarded by very
hot, dry weather.
15. Penetration of the host is by hyphae of germinated spores enter
ing stomata of the leaves.
16. Pathological anatomy is described in detail. The mycelium of
each of the causal organisms grows intercellularly and does not
produce haustoria.


Fig. 3.--Lesions on old chrysanthemum leaves
6 weeks after inoculation with spores of Septoria
chrysanthemella (X 1/3).


4
states were examined. The Septoria obesa disease was found on collec
tions from 13 of these states, whereas the Septoria chrysanthemella
disease was found on specimens only from Iowa. Texas, and Tennessee.
Economic Importance
The chrysanthemum is one of the most important flowers of
commerce in the United States and also in several other parts of the
world. Most of the plants are ultimately sold as cut flowers, but there
is also a large trade in potted plants and in rooted cuttings for plant
ing in private gardens.
The septoria leafspot diseases were very severe in commercial
beds in the United States until World War II. The diseases are now
controlled to a great extent in commercial plantings by dithiocarbamate
sprays but frequently become severe during extended rainy periods. The
diseases remain a problem in private gardens where control measures are
usually less rigidly practiced.
The writer agrees with the statements of Dimock (10) concerning
losses due to septoria leafspots:
1. Spotting of foliage seriously detracts from the
quality and salability of cut flowers.
2. Even if spotting is confined to the lower leaves,
the loss of lower foliage may seriously reduce both
the quantity and quality of blooms.
As previously stated, the disease caused by Septoria obesa is
apparently more common than the Septoria chrysanthemella disease in the
United States, and. as will be described later, it is considerably more
severe,


Fig. 8;Longitudinal section of a pycnidium
of Septoria chrysantheaella in chrysanthemum leaf
tissues (X476).


5
Symptoms
The following descriptions are based on observations of the
natural development of the diseases in the field and greenhouse and on
inoculation experiments under various controlled conditions*
DESCRIPTIONS.The disease caused by Septoria obesa is usually
considerably more severe than the disease caused by Septoria chrysanthe-
mella. Lesions begin as small chlorotic areas which usually become
dark brown or black and enlarge to form circular to very irregular areas
with distinct margins* Frequently* however* the young lesion develops
into a blackish or brownish blotch-like lesion of indefinite size and
shape with indistinct margin. The latter type of lesion bocones black
and the margin becomes distinct during rainy weather* A yellow halo
usually surrounds the lesion* A single lesion may spread to occupy
1/3 or more of a leaf* and a leaf may be killed by one or two lesions*
The lesions are distinct on both surfaces of the leaf but are darker
cm the adaxial surface. Pycnidia are usually abundant cm both surfaces
of the lesions*
The disease attacks and kills the lower leaves first and under
favorable conditions progresses upward* finally attacking the upper
leaves and the lnvolucral bracts* Entire shoots or entire plants are
frequently killed* The symptoms are shown in Figs* 1 and 2*
The disease caused by Sep tori a chrysanthemell a occurs cm
susceptible chrysanthemum plants of all ages* The first symptom is the
appearance of very small chlorotic spots* barely discernible without
magnificat!cm* cm the leaf blades* Under favorable moisture and humidity


16
Morphology
Both Septoria obesa and Septoria chrysanthemella produce
nycelia, pycnidia, and pycnidiospores in leaf tissues of susceptible
hosts and in suitable artificial culture media. A perfect stage of
either of these fungi has not been reported. Voglino (38) in 1901
reported what he termed a phoma stage of S. chrysanthemella on the
leaves and stems of chrysanthemums. This stage has not been observed
by any other investigator, and it seems likely that it was produced
by a fungus distinct from S. chrysanthemella.
Several authors have given descriptions of the pycnidia and
pycnidiospores of Septoria obesa and Septoria chrysanthemella. A
summary of these descriptions is given in Tables 1 and 2.
TABLE! 1
MORPHOLOGICAL CHARACTERS OF SEPTORIA OBESA AS REPORTED
BY DIFFERENT INVESTIGATORS
Investigators
Morphological
Character
H. & P.
Sydow (36)
Hemrni &
Nakamura (22)
Loos (24)
Diameter of pycnidia
120-160 u
75-200 u
87-173 u
Length of pycnidiospores
50-100, rarely 41-124 u
120 u
66-87 u
Width of pycnidiospores
3-4.5 u
2.5-4.4 u
3.2-3.6 u
Number of septa
5-12
4-12
up to 9
Shape of pycnidiospores
narrowly
obclavate
whip-shaped
Rounded at
base, tapered
toward apex


DISCUSSION.Cavara (32) described the pycnidiospores of
Septoria chrysanthemella as being non-septate; however, the septa in
this species are usually invisible unless the spore walls are artificially
stained. Hemal and Nakamura (22) describe the pycnidiospores of Septoria
obesa as being obscurely septate, and these investigators used an iodine
stain in order to observe the septa. The pycnidiospores of S. obesa
observed by the writer were all distinctly septate if well matured. It
is possible that Hemmi and Nakamura observed the spores just prior to
their full maturity, or there may be a difference in the distinctness of
septations in the species in different parts of the world or under dif
ferent environmental conditions.
The two species of Sentoria on the chrysanthemum collected in
the United States are Septoria chrysaathemella and Septoria obesa.
There was no significant difference in the mean length of either
Septoria obesa pycnidiospores or Septoria chrysaathemella pycnidiospores
produced on six different chrysanthemum varieties. It cannot be stated
that the host variety does not affect this character, as a comparatively
small number of varieties was used, and they were not selected at random
from the several thousand varieties in cultivation.
No significant difference was found in the mean length of either
Septoria obesa or Septoria chrysanthemella pycnidiospores collected from
different locations in the United States. Geographical locations were
not randomly selected. Pycnidiospore length could possibly be affected
by both environmental conditions and hereditary factors.
There was a considerable difference between the dimensions and
number of septa of pycnidiospores and also between the pycnidial


22
Fig. 6.Pycaidiospores of Sep tori a obesa (X 47G)


40
tabu; s
THE EFFECT OF HYDROGEN-1 ON CQNCENTRATI ON ON THE GROTTO OF
SEPTORIA OBESA IN A SYNTHETIC LIQUID MEDIUM
pH
Dry Mycelial Weight
Range
Mean
Mean
Final pH
2
no growth occurred
3
45.0-57.9
53.4
3.3
4
41.6-81.7
54.6
4.5
5
98.0-169.5
129.6
6.9
6
109.8-133.7
123.9
7.1
7
73.4-125.2
99.0
7.2
8
46.0-71.1
60.2
7.7
9
31.4-40.0
36.2
8.6
10
no growth occurred
sl
After 28 days of growth,
b
Six replications were used.


37
Pathogenicity
In numerous inoculation experimente all isolates of Septoria
obesa and all isolates of Septoria chrysanthemella produced character
istic disease symptoms on certain chrysanthemum varieties, whereas
check plants remained healthy. In all cases, pycnidiospores character
istic of the Septoria species involved were abundant in the disease
lesions of the host foliage.
It has been reported by Gram and Weber (17), Raff (30), Bohmig
(3), and Fischer (16) that chrysanthemum varieties vary in suscepti
bility to septoria leafspot. However, all of these reports were based
on observations and none were made under controlled conditions by
artificial inoculations.
The pathogenicity of Septoria obesa and Septoria chrysanthe-
mslla on twenty-two varieties of chrysanthemum were investigated in
a series of inoculation experiments conducted under variable conditions
in the greenhouse and in the field.
METHODS AND MATERIALS.All plants in the greenhouse were grown
in 4-inch, clay pots containing soil which had previously been dis
infected with methyl bromide. Hie soil in outdoor plots was not treated;
however, no chrysanthemum had been grown in the immediate vicinity for
several years. Fungicides were not used on experimental plants, but
propagation stock was treated with a ferbam spray about once a week.
Malathioa or nicotine sulfate sprays were used as needed for insect
control on all plants


This dissertation was prepared under the direction of
the chairman of the candidate's supervisory committee and has
been approved by all members of that committee. It was sub
mitted to the Dean of the College of Agriculture and to the
Graduate Council, and was approved as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.
August S, 1959
OaaA. Dean, College of Agriculture
Dean, Graduate School
SUPERVISORY COMMITTEE:


70
day temperatures in the greenhouse during sunny summer days usually
ranged between 85 and 95F. During mild weather symptoms of both
diseases usually become visible in 12 to 18 days and pycnidiospores
are present about 1 week later* This agrees with Dimock (10) who
gives the incubation period at 10 to 14 days, with spores developing
4 to 6 days later.
It was noted by Halsted (20) in 1891 that moisture greatly
favors the development of septoria leafspots, and this has been
frequently observed since that time. Not only are the spores dis
seminated during rainy periods, causing new infections, but lesions
already present increase in size.
Host Penetration
According to Dimock (10, 12) infection by Septoria obesa or
Septoria chrysaathemella occurs almost exclusively at the abaxial sur
faces of the leaves. In several inoculation experiments carried out
with plants under bell jars over water* the writer found this to be
true; only rarely did infection occur when only the adaxial surfaces
of leaves were inoculated. However, literature reveals no information
on the specific mode of host penetration by either fungus; consequently,
a study of this was undertaken*
METHODS AND MATERIALS.After much experimentation with various
leaf-clearing and staining techniques, the best method, and also the
most rapid, was found to be a combination of the method used by Peace
(29) to clear dead leaves and a technique described by Diener (9) for
staining fungus spores and hyphae on leaves.


TABUS 3
DIMENSIONS AND NUMBER OF SEPTA OF PYCNIDIOSPORES AND DIAMETERS OF
FYCNIDIA OF SEPTORIA OBESA SPECIMENS COLLECTED AT
VARIOUS LOCATIONS IN TEE UNITED STATES
Pycnidiospore Dimensions
Microns
Pycnidiospore
Septations
Pycnidia Diameter!
Microns
Specimen
No.a
Length
Range Meanb
Width
Range
Range
Range
1
54-106
78.0
2.8-4.5
7-13
80-156
2
46-104
75.5
2.6-4.0
7-14
74-148
3
45-100
73.2
2.8-4.5
6-13
62-156
4
44-102
72.9
2.7-4.4
6-14
60-123
5
44-100
71.4
2.8-4.4
5-14
75-132
6
50-108
74.7
2.7-4.5
6-14
68-160
7
45-98
74.2
2.6-3.9
5-13
72-149
8
45-100
73.8
2.7-4.3
7-14
70-140
Overall
44-108
74.2
2.6-4.5
5-14
60-160
aThe sample size was 34 pycnidiospores or pycnidia.
An analysis of variance gave no significant difference in the
mean length of pycnidiospores from different specimens.


42
The amount of growth of Septoria obesa in synthetic liquid
media and on semi-synthetic agar medium at various temperatures is
shown in Tables 10 and 11, respectively. The results of similar experi
ments with Septoria chrysanthemella are shown in Tables 12 and 13.
S. obesa and S. chrysanthemella in culture on semi-synthetic agar
medium at optimum temperatures are shown in Fig. 12.
TABLE 10
THE EFFECT OF TEMPERATURE ON THE GROWTH OF SEPTORIA OBESA
IN SYNTHETIC LIQUID MEDIA
c
Dry Mycelial Weight
Range
in mga
Meanb
4
33.6-42.2
39.0
8
69.2-78.5
73.9
f O
102.0-114.2
109.5
16
12S.4-136.4
132.3
20
118.2-131.4
125.0
24
80.2-88.5
64.9
28
25.4-30.2
27.7
32
no growth occurred
aAfter 28 days of growth.
i-
Five replications were used.


44
TABLE 12
TOE EFFECT OF TEMPERATURE ON THE GROWTH OF SEPTORIA
CHRYSANTIEMELLA IN SYNTHETIC LIQUID MEDIUM
Dry Mycelial Weight in mga
c
Range
Mean0
4
22.4-25.0
23.7
8
61.2-62.8
62.0
12
94.0-102.2
97.6
16' 1
105.7-121.4
113.1
20
93.4-109.0
100.6
24
71.8-78.6
75.6
28
61.0-64.5
62.7
32
no growth occurred
aAftor 28 days of growth.
|j
Three replications were used.


14
to read Joffrin's description of this fungus, but as described by
Chifflot the pycnidiospores are filiform and many-celled, and figures
of the spores shoe than to be obclavate and 10-14 septate* If
Chifflot's description is accurate, Septoria varians is not the same
as Septoria chrysanthemella.
The valid name and synonyms of the fungus under consideration
are as follows:
Septoria chrysanthemella Sacc. (sub nom. Septoria
chrysanthemella Cav* 1895)
Synonyms:
Septoria chrysanthemi Cav* 1892*
Septoria chrysanthemi Rostr* 1897*
Septoria rostrupii Sacc. & Syd* 1899*
SEPTORIA OBESA SYD.*In 1914 H. and P* Sydow (36) described a
new fungus, Septoria Obesa Syd., parasitic on Chrysanthemum arcticum,
a wild plant of northern Japan.
In 1917 Hemmi (22) found too different septorias parasitic on
Chrysanthemum morifolium in Japan. One species was identified as
Septoria chrysanthemella. The other species was first identified as
Septoria chrysantheml-indici Bub. & Kab., but after examining the type
specimen of this fungus, Hemmi reversed his opinion. Subsequently
Hemmi studied Sydow*s description of Septoria obesa and examined the
type specimen. He found that his species was morphologically identical
to Septoria obesa Syd. and stated that the two are synonymous. Theo
retically, Hemmi's species should have been described as a new species
or as a variety of S. obesa. The two occurred on different hosts and
cross-inoculation experiments were not conducted.


45
TABUS 13
THE EFFECT OF TEMPERATURE OH THE GROWTH OF SEPTORXA
CHRYSANTHEMELLA OH SEMI-SYNTHETIC AGAR 1KDIIB!
c
Culture Diameters
Range
in mma
Meaab
Sporulation
4
1.5-3.0
2.3
no
8
5.5-7.0
6.1
yes
12
10.5-12.0
11.3
yes
16
16.0-19.0
17.0
yes
20
23.5-28.5
26.0
yes
24
25.0-30.0
27.6
yes
28* '
31.5-35.5
33.5
yes
32 no growth occurred
aAfter 28 days of growth,
b
Four replications were used.


43
TABUS 11
THE EFFECT OF TEMPERATURE ON THE GROWTH OF SEPTORIA OBESA
ON SEMI-SYNTHETIC AGAR MEDItBS
c
Culture Diameter
Range
in mma
Moan'0
Sporulation
4
2.5-3.5
3.0
no
8
5.56.5
6.0
yes
12
7.0-8.0
7.4
yes
16
10.5-12.0
11.0
yes
20
12.0-14.5
13.4
yes
24 V 'f
15.0-16.5
15.8
yes
28
12.2-14.4
13.2
yes
32 no growth occurred
aAfter 28 days of growth,
b
Four replications were used.


63
TABLE 21
SEVERITY OF DISEASE ON TWENTY-TWO CHRYSANTHEMUM VARIETIES INOCULATED
WITH SEPTORIA CHRYSANTI IE MELLA IN THE GREENHOUSE
Chrysanthemum No. of No. of Plants in Each Disease Class
Variety Plants Severe Moderate 1 Mild Healthy
Alaska 8
Beauregard 8
Blue Chip 8
Bonnie 8
Copperhead 8
Dark Buckley 8
Delaware 8
Gold Ball 8
Humdinger 8
Illini Igloo 8
Illini Regal 8
Illini Warpaint 8
Indianapolis Bronze 8
Jetfire 8
Mary L. Hall 8
Pink Dot 8
Portrait 8
Rayonnaate 8
Seneca 8
White Top 8
Yellow Queen 8
Yellow Shasta 8
Control Plants 44
1
7
8
1
5
4
2
1
6
3
1
1
6
1
7
2
3
4
5
3
2
4
5
4
4
2
4
3
6
3
2
2
6
4
1
2
3
5
1
3
4
4
6
3
5
2
4
5
2
4
44


39
previously described after Incubation of the cultures for 28 days at
20C.
The effects of several nitrogen sources on the growth of the
fungi in culture were studied. Semi-synthetic agar medium less biotin
and thiamine and with other nitrogen sources alternated with L-asparagine
was used. The quantity of each nitrogen source used was adjusted to
give a concentration of 425 mg of nitrogen per liter. The compounds
and quantities used per liter are given below:
L-asparagine 2.000 g
Glycine 2.277 g
L-leucine 3.977 g
L-arginine 1.321 g
Ammonium sulfate 2.014 g
Potassium nitrate 3.066 g
Controlnltrogen compounds omitted
Four replications for each fungus were used. The pH was ad
justed to 5.0 after autoclaving. Incubation was for 28 days at 20C.
Germination studies were made on pycnidiospores produced in
potato-dextrose-agar medium. The percentage of germination on water
agar at various temperatures was determined by observing 50 spores at
each temperature. Observations on pycnidiospore germination in several
kinds tt media were made.
RESULTS.The optimum pH for the growth of both Septoria obesa
and Septoria chrysanthemella in synthetic liquid medium was pH 5 to
pH 7. No growth occurred at pH 2 or pH 10, and little occurred at
pH 3 or pH 9. Summaries of the growth of S. obesa and S. chrysanthemel1a
are given in Tables 8 and 9y respectively.


TABLE 6
COMPARISON OF PYCNIDIOS PORE DIMENSIONS ON SIX CHRYSANTHEMUM VARIETIES
INOCULATED WITH ISOLATE NO. 1 OF SEPTORIA CHRYSANTHEMELLA
Chrysanthemum Variety^
Range
Length in Microns
Mean11
Alaska
30-68
38.4
Bonnie
29-72
40.8
Delaware
28-72
39.7
Mary L. Hall
32-74
41.5
Rayonnante
28-58
37.7
Yellow Shasta
25-68
37.2
Overall
25-74
38.9
aThirty-four pycnidiospores were measured in each variety,
b
There was no significant difference in mean length of spores
among varieties at the 5 per cent level.


LIST OF TABLES (continued)
Table Page
13.The Effect of Temperature on the Growth of Septoria
chrysanthemella on Semi-synthetic Agar Medium .... 45
14.The Effect of Carbon Source on the Growth of
Septoria obesa on Semi-synthetic Agar Medium .... 47
15.The Effect of Carbon Source on the Growth of
Septoria chrysanthemella on Semi-synthetic Agar Medium 48
16,The Effect of Nitrogen Source on the Growth of
Septoria obesa on Semi-synthetic Agar Medium .... 49
17. The Effect of Nitrogen Source on the Growth of
Septoria chrysanthemella on Semi-synthetic Agar
Medium ........ 50
18. Percentage of Germination of Pycnidiospores on Water
Agar at Various Temperatures ..... 54
19. Severity of Disease on Mature Plants of Fifteen
Chrysanthemum Varieties Inoculated with Septoria obesa
in the Field 61
20. Severity of Disease on Twenty-two Chrysanthemum
Varieties Inoculated with Septoria obesa in the Green
house 62
21. Severity of Disease on Twenty-two Chrysanthemum
Varieties Inoculated with Septoria chrysanthemella
in the Greenhouse ............ 63
22. Severity of Disease on Fourteen Chrysanthemum
Varieties Inoculated with Septoria chrysanthemella
in the Field 64
v


LIST OF FIGURES (continued)
Figure Page
14, Pycnidiospores of Septoria chrysanthemel1a Germinat
ing on Solid Medium .......... 53
15, Pycnidiospores of Septoria chrysanthemella Germinat
ing in Water 53
16, Stomatal Penetration by a Hypha from a Germinating
Pycni dio spore of Septoria obesa 73
17. Stomatal Penetration by a Hypha from a Germinating
Pycnidiospore of Septoria chrysanthemella 73
13. Cross-section of Host Leaf Shoving Intercellular
Invasion of Mesophyll Tissue by Septoria obesa
Mycelium ... 75
19. Cross-section through a Leaf Lesion Showing Pycnidia
of Septoria obesa 76
20. Cross-section through a Leaf Lesion Shoving Pycnidia
of Septoria chrysanthemella 76
vil


SUMMARY
1* Two distinct septoria diseases of the cultivated chrysanthemum
are common in the United States and in several other parts of
the world where the host is grown.
i
2. The valid names of the causal organisms are Septoria obesa Syd.
and Septoria chrysanthemella Sacc.
3. The disease caused by Septoria obesa appears to be more common
in the United States and is also more severe than the disease
caused by Septoria chrysanthemella.
4. Symptoms of each disease are described in detail. Septoria obesa
caused black or dark brown leaf lesions which are circular to
irregular in shape and indefinite in size. Septoria chrysanthe
mella causes black, circular leaf lesions which are usually one
centimeter or less in diameter.
5. Hie morphology of the causal organisms in culture and in host
tissues is described in detail. There is a distinct difference
in average size, shape, and number of septa of the pycnidiospores
of the two pathogens. Geographical location or host variety did
not significantly affect pycnidiospore morphology.
6. The optimum hydrogen-ion concentration for the growth of each
fungus in culture was pH 5 to pH 7; no growth occurred at pH 2
or pH 10.
7. The optimum temperatures for the growth of each pathogen in
culture on solid medium was 20 to 28C. Little growth occurred
at 4C., and there was no growth at 32C.
8. The rate and character of growth of the pathogens in culture on
various carbon sources and nitrogen sources is described.
9. Pycnidiospores germinated rapidly on solid media and germinated
slowly in water. The rate of germination at various temperatures
is described.
10.Septoria obesa severely attacked all chrysanthemum varieties
inoculated, but the pathogenicity of Septoria chrysanthemella
varied greatly on different varieties of the host.
77


Fig. 16.Stomatal penetration by a hypha from
a germinating pycnidiospore of Septoria obesa (X 476)
Fig. 17.Stomatal penetration by a hypha from
a germinating pycnidiospore of Septoria chrysanthemella
(X 476).


21
Fig, 5.Longitudinal section of a pycnidium of
Septoria obesa in chrysanthemum leaf tissues (X 476).


so
with small whitish areas dispersed near their centers. With L-leucine
a white culture with dispersed black spots resulted and ammonium sulfate
gave a white fluffy culture with a greenish center. Cultures which
lacked nitrogen had yellow interiors which were covered by a white
mycelium. No pycnidiospores were produced when ammonium sulfate was
used or when nitrogen was lacking, and very few were formed when glycine
was used. Cultures of the fungus growing on different nitrogen sources
are shown in Fig. 13.
TABUS 17
the effect of nitrogen source on the growth of septoria
CHRYSANTHEMELLA ON SEMI-SYNTHETIC AGAR MEDIUM
Nitrogen
Source*5
Culture Diameter
Range
in mma
Meanc
Sporulation
Glycine
14.5-18.5
15.8
Very little
L-asparagine
32.5-33.5
33.3
Yes
L-leucine
20.0-22.0
21.0
Yes
L-arginine
28.0-27.0
26.6
Yes
Ammonium sulfate
17.0-21.5
18.9
No
Potassium nitrate
19.5-24.0
22.0
Yes
Minus nitrogen
15.0-16.5
15.6
No
a
After 28 days of growth,
b
All nitrogen sources were adjusted to give 425 mg of nitrogen
per liter,
c
Four replications were used.


At temperatures of 4 to 20%. in the liquid medium, both of
the fungi produced black or greenish-black, compact masses of mycelium
which tended to adhere to the bottoms or sides of the vessels. At
24 and 23C. a colorless, film-like mycelium was formed which tended
to float on the surface of the liquid.
Temperature had no apparent effect on the color of cultures of
either fungus grown on the solid medium.
Both Septoria obesa and Septoria chrysanthemella sporulated on
all carbon sources used and also in the absence of carbon sources.
Data chi culture diameters are given in Tables 14 and 15.
TABLE 14
THE EFFECT OF CARRON SOURCE ON THE GROWTH OF SEPTORIA
OBESA ON SEMI-SYNTHETIC AGAR MEDIUM
Carbon
Culture Diameter
in mma
Source**
Range
Mean*3
Sporulation
Mannose
7.0-12.0
10.1
Yes
D-mannitol
7.0-8.5
8.0
Yes
Maltose
8.5-10.5
9.7
Yes
Galactose
8.5-11.5
10.5
Yes
Sucrose
9.0-11.5
9.9
Yes
Dextrose
10.0-11.0
10.4
Yes
Lactose
5.5-8.0
6.9
Yes
Minus carbon source
3.5-7.0
6.0
Yes
a
After 28 days of growth.
bAt the rate of 10 g per liter.
Four replications were used.


inoculated with 3-day old germinating pycnidiospores and incubated for
28 days at 20C. Dry mycelial weights were then determined. Four
replications were used for Septoria chrysanthemella and 6 for Septoria
38
obesa. The experiments were not conducted concurrently for the two
species of Septoria, but all conditions were duplicated as nearly as
possible.
The effects of different temperatures on the growth of the fungi
in both synthetic liquid medium and semi-synthetic agar medium were
investigated. Germinating spores in 125 ml portions of the liquid
medium were incubated for 28 days at four-degree intervals from 4 to
32C., and mycelial weights were then determined. Three replications
were used for Septoria chrysanthemella and 5 for Septoria obesa. In
festation of the solid medium in petri dishes was accomplished by first
inserting a sterile needle into a speculating culture and then inserting
the needle vertically into the center of the medium. These cultures
were incubated for 2 days at 20C. and were then transferred to constant
temperature chambers at the temperatures given above, where they remained
for 28 days. The average diameter of each culture was then estimated by
making two measurements through the center of the culture. Four repli
cations of each fungus were used in the experiments.
The effects of different carbon sources on the growth of the
fungi on semi-synthetic agar medium was observed. Carbon sources used
in the experiments were mannose, D-mannitol, maltose, galactose, sucrose,
dextrose, and lactose; in addition there was a control group which lacked
a carbon source. All carbon sources were used at the rate of 10 g per
liter. The pH was adjusted to 5.0 after autoclaving. Four replications
were used for each fungus. The mean diameters were estimated as


68
greenhouse bench. The same procedure was followed, and at the same time,
with Septoria chrysanthemella except that the chrysanthemum variety used
was Mary L. Hall.
All of the plants inoculated with the suspension containing the
diseased leaf fragments became infected. Disease lesions were not
numerous on these plants, but pycnidiospores characteristic of the species
of Septoria involved were eventually produced. None of the plants dipped
into the suspension of field soil became infected. All plants inoculated
with spore suspensions from culture became heavily infected.
On April 5 no spores could be found in the over-wintered inoculum.
However, the inoculation experiment as described above was repeated, but
the only plants that became infected were those inoculated with spores
from culture.
It appears to be very doubtful that inoculum in diseased leaves
left on the soil in the fall remains viable into the late spring or into
the summer in the Gainesville area.
After plants have become infected, pycnidia and pycnidiospores
are formed in the leaf lesions. The pycnidiospores produce secondary
infection when disseminated to other leaves or other plants if conditions
are favorable.
Spore Dissemination
It has been noted by many observers that new infections of
chrysanthemum plants by septoria diseases follow periods of rainy
weather. According to Dimock (10) the pycnidiospores are disseminated
by splashed or wind-blown water, or by various mechanical means when


Fig. 11.--Camera lucida drawing of pycnidio-
spores of Septoria chrysanthemella (left)and
Septoria obesa (right).


7
lack or have only a few pycnidia on their ahaxial surfaces*
Young lesions produced by Septoria obesa are frequently small
and circular, thus resembling those of Septoria chrysanthemella* The
two diseases can be distinguished with certainty by observing the
pycnldiospores produced in the lesions. The morphology of the pycnidio-
spores will be described later.


49
TABLE 16
THE EFFECT OF NITROGEN SOURCE ON THE GROWTH OF SEPTQRIA OBESA
ON SEMI-SYNTHETIC AGAR MEDIUM
Nitrogen
Source*1
Culture Diameter
Range
in imna
Mean0
Sporulation
Glycine
4.5-6.0
5.1
Yes
L-asparagine
11.5-13.5
12.8
Yes
L-leucine
4.5-6.0
5*0
Yes
L-arginine
8.09.5
8.9
Yes
Ammonium sulfate
7.0-3.0
7.5
Yes
Potassium nitrate
7.0-11.0
8.3
Yes
Minus nitrogen
5.56.5
6.1
Yes
aAfter 28 days of growth.
bAll nitrogen sources wore adjusted to give 425 ng of nitrogen
per liter.
CFour replications were used.
Septoria chrysanthcmsl1a grown on artificial media containing
different nitrogen sources varied markedly in both size and color. The
diameters of the cultures are given in Table 17. When L-asparagine was
used as a nitrogen source the culture was black, compact and raised, as
is typical of this fungus grown on potato-dextrose-agar. With glycine,
the cultures were conspicuously concentrically zoned with narrow dark
and light areas; zonation also occurred when L-arginine was used but was
not as distinct as with glycine. Potassium nitrate cultures were black


the foliage is wet. He found that direct splashing of spores from the
soil may reach a height of 16 to 18 inches. Dimock (11) also found
that the spores were disseminated by insecticidal sprays and that most
insecticides commonly used have little or no effect on the viability
of the spores.
Climatic Conditions Affecting Infection and Disease Development
According to Dimock (10), pycnidiospores must be in contact
with the wet abaxial surface of a leaf for at least 24 hours before
infection is established; the 24-hour wet period does not have to be
continuous for infection to occur but may alternate with dry periods
such as may occur with nightly dews and sunny days.
Hie writer agrees that some infection by each of the Septoria
species may occur after a 24-hour wet period for the pycnidiospores on
the abaxial surface of the leaf. However, several experiments in which
inoculated plants were maintained in a mist chamber for only one day
resulted in little or no infection. Furthermore, only an occasional
germinated spore of either fungus was observed on cleared leaves which
had previously been inoculated and kept wet for 24 hours at 20C; even
after 43 hours only about one-half of the spores had germinated.
Extremely hot weather retards the development of the diseases,
both in the field and the greenhouse. All inoculation experiments
carried out in the greenhouse during the hot summer months either failed
completely, or else the development of symptoms was arrested until a
period of mild weather arrived. In one case plants inoculated with
Septoria obesa did not show disease symptoms until 6 weeks later. The


41
TABLE 9
THE EFFECT OF HYDROGEN-ION CONCENTRATION ON THE GROWTH OF
SEPTOP.IA CIIRYSANTIIEMELLA IN A SYNTHETIC LIQUID MEDIUM
Dry Mycelial Weight in mga .
Mean
pH
Range
Mean
Final pH
2
no growth occurred
3
36.0-44.2
40.0
3.4
4
47.0-56.6
50.9
4.5
5
92.8-105.0
99.2
6.2
6
99.2-111.3
105.2
6.9
7
89.6-100.0
95.6
7.0
8
43.0-55.4
49.0
7.7
9
26.0-35.6
31.2
8.6
10
no growth occurred
aAfter 28 clays of growth,
b
Six replications wore used.


Fig. 4.--Lesions on y e're.yr.aathemuin leaves
8 weeks after inoculation with spores of Septoria
chrvsanthemel1a (X 3/4).


28
Summaries o pycnidial diameters and pycnidiospore dimensions
and number of septa observed in leaf specimens collected in various
locations in the United States are given in Tables 3 and 4 for Septoria
obesa and Septoria chrysanthemella,respectively. Similar information
is given in Tables 5 and 6 for pycnidiospores produced in different
*
chrysanthemum varieties after artificial inoculations with isolate No. 1
of S. obesa or isolate No. 1 of S. chrysanthemella, respectively.


6
conditions, these spots bee cm black in a few days and gradually en
large to form black, circular or elliptical spots, mostly 1 cm or less
in diameter but sometimes as large as 2 cm in diameter. These lesions
usually show distinct margins, and they are sometimes surrounded by
narrow chlorotic halos. They are visible on both surfaces of the leaf
but are considerably more conspicuous on the adaxial surface.
When the adaxial surface of a lesion is examined under a hand
lens, numerous black pycnidia are seen embedded in the leaf tissue.
There may be only a few lesions on an infected leaf, or there may be
so many that they coalesce, thus forming large necrotic areas. The
centers of old lesions sometimes become grayish in color.
The lower leaves of a plant are infected first, and if infection
is severe they die and hang downward on the stem. Following periods of
rainfall, the disease progresses upward on the plant, and the uppermost
leaves and the involucra! bracts may eventually become infected.
Symptoms of this disease are shown in Figs. 3 and 4.
COMPARISON.Advanced symptoms produced by Septoria obesa under
favorable conditions of moisture and temperature are distinctly dif
ferent from those produced by Septoria chrysanthemella, the leaf lesions
being large and circular to irregular in shape. S>. chrysanthemella
causes circular to elliptical leaf lesions which usually do not exceed
1 cm in diameter, but these lesions may coalesce to form black, irregular
shaped, necrotic areas; however, when closely observed, the individual
spots making up the irregular areas are easily discerned. Another point
of difference is the occurrence of abundant pycnidia chi the abaxial
surface of S. obesa lesions, whereas S. chrysanthemella lesions either


74
the host cells and causing their death. No evidoace of direct
penetration or haustoria formation was observed. The mycelium grows
most freely in the spongy and palisade mesophyll but also invades the
bundle sheaths and vascular tissues. The mycelium was observed to
extend as much as 2 mm from the necrotic tissue of the lesion.
Figure 18 shows the mycelium of Septoria obesa surrounding cells of
the mesophyll. *
Hie mycelium surrounding lysigenous cavities adjacent to
epidermal tissues sometimes forms numerous small, spherical or
elliptical-shaped cells. Hiis is apparently an early stage in the
formation of a pycnidium. Pycnidia were present only in necrotic
tissue in this material but have been observed in adjacent chlorotic
tissues in untreated leaves. Short pycnidial beaks rupture the epidermis
and extend above it or sometimes extend through stomata.
Host tissue invaded by Septoria obesa and Septoria chrysanthemella
are shown in Figs. 19 and 20, respectively.


23


HOST-PARASITE RELATIONS
Seasonal Development of the Diseases
The development of the disease caused by Septoria obesa has
been observed for the past 13 months in unprotected chrysanthemum
plantings in Gainesville, Florida. In a field plot on which chrysan
themums were heavily diseased the previous fall, young shoots developed
symptoms about two weeks after emerging from the soil in February and
March.
Once infection has occurred, the plants remain diseased until
killed by freezes the following winter. The development of symptoms
and the spread of the disease are affected more by climatic conditions
than by the season; however, in general, the development of symptoms
and the spread of the disease are more rapid in the spring and fall
than in the summer.
The seasonal development of the disease caused by Septoria
chrysanthemella was not observed, but various reports in literature
indicate that its seasonal development is very similar to that of
Septoria obesa.
Sources of the Inoculum
The source of primary inoculum in the field is the pycnidio-
spores in pycnidia imbedded in diseased leaves remaining on the ground
in the fall and winter when plants are not growing. Dimock and Allen
(13) observed the disease develop late in the summer in cut-over beds
where septoria leafspot had been controlled during the earlier part of
66


24
Septoria chrysanthemella.Pycnidia formed in host tissues
(Fig. g) are mostly pyriform but frequently globose or subglobose in
shape, 40-124 microns in diameter, and slightly to 1/2 erumpent.
Ostioles occur at the ends of short to rather elongate beaks and vary
in diameter from being almost closed to being about 1/2 or more the
diameter of the pycnidiurn when spores are being exuded. Hie walls of
the pycnidia are composed of 3 to 6 layers of compact cells which are
usually nearly isodiametric but sometimes 3 or 4 times as long as broad.
%
Hie outer cell layers are brown in color but the innermost layer is
hyaline. Conidiophores are composed of the inner cells of the pycnidial
wall which have elongated slightly toward the center of the pycnidium.
They are hyaline and 3 to 6 microns in length. Pycnidiospores (Fig. t)>
are filiform, usually straight but sometimes slightly curved, uniform
in width and bluntly pointed at base and apex, obscurely 0-5 septate,
22-70 x 1.8-3 microns, hyaline, finely granular, and usually several
guttulate. The mycelium is similar to that described above for Septoria
obesa.
In culture on potato-dextrose-agar medium, the morphology of
the pycnidiospores of Septoria chrysanthemella is as described above.
Pycnidia in culture are globose to very irregular in shape and the walls
are usually much thicker than in host tissue. The first pycnidia formed
are almost superficial on the surface of the culture and are oriented
with their ostioles communicating with the internal portion of the
culture (Fig. 10). Only occasionally do ostioles open on the surface.
Pycnidia formed in deeper layers of the culture are oriented variously.
Hie morphology of the mycelium is similar to that described for Septoria
obesa,


ACKNOWLEDGMENTS
The writer greatly appreciates the guidance, helpful sug
gestions, and aid in manuscript preparation given to him by Dr.
George F. Weber, chairman of his supervisory committee. Appreciation
is expressed to the other members of the supervisory committee
Dr. Mildred Griffith, Dr. Phares Decker, Dr. P. H. Senn, and Prof.
Albert Mullerfor their help and consideration.
Acknowledgments are gratefully made to the University of
Florida Graduate Council, the Southern Fellowships Fund, and the
National Science Foundation for fellowships without which this work
could not have been completed.
Sincere gratitude is expressed to Myra, the devoted wife of
the writer, for encouragement and for the performance of innumerable
tasks, including the typing of this manuscript.
ii
V / (? 51


46
Fig. 12.Growth of Septoria obesa (left) and
Septoria chrysanthemella (right) on semi-synthetic
agar medium at optimum temperatures at the end of
40 days.


53
Fig. 14.Pycnidiospores of Septoria ehrysanthe-
mella germinating on solid medium Fig. 15.Pycnidiospores of Septoria chrysanthe
me! la germinating in water (X 1000).


DISCUSSION.The optimum pH for the growth of both Septoria
obesa and Septoria chrysanthemella was found to be from pH 5 to pH 7, \D
These findings are not exceptional, as most fungi which have been
investigated grow best in slightly acid media.
Both fungi made relatively poor growth in liquid medium at 24 ^
and 28C. whereas maximum growth on solid medium was at these same
temperatures. The poor growth at the higher temperatures in liquid
medium could possibly be related to the lower retention of oxygen and
other gases by liquids at higher temperatures, or chemical changes un
favorable to the fungi may have occurred at higher temperatures. Neither
fungus grew at 32C. With regard to Septoria chrysanthemella, this is
in conflict with the findings of Heneni and Nakamura (22) that this
fungus made better growth at 32C. than at 20C. This difference could
be due to experimental errors or to slight physiological differences
between the American isolates and the Japanese isolates.
Each of the two fungi grew and sporulated on all carbon sources
used and also in the absence of a carbon source. Growth of both of the
species was poorest when carbon was lacking and when lactose was used
as a carbon source. There was apparently very little difference in the
rate of growth of either of the fungi on the several other carbon sources
used.
Both Septoria obesa and Septoria chrysanthemella grew more
rapidly when L-asparagine was used as a nitrogen source than when other
nitrogen sources were used. The growth of both fungi was relatively slow
on glycine, ammonium sulfate, and in the absence of nitrogen. Septoria


81
31.
Saccardo,
P.
A.
1884.
Sylloge
fungorum 3:549.
32.
Saccardo,
P.
A.
1895.
Sylloge
fungorum 11:542.
33.
Saccardo,
P.
A.
1898.
Sylloge
fungorum 14:973.
34.
Saccardo,
P.
A.
1913.
Sylloge
fungorum 22:1104.
35.
Salmon, E. S
(Abs.) Rev.
, and W. M. Ware. 1937. Department of mycology.
Appl. Mycol. 16:366.
36.
Sydow, H.
and P.
Sydov.
1914.
Zweiter Beitrag zur Kenntnis der
parasitischen Pilzflora des nordlich Japans. Asnales Mycologici
12:184.
37. Trent, J. A. 1939. The status of Cylindrosporium chrysanthemi
S. & D. as the causative agent of chrysanthemum leaf blight.
Trans. Kans. Acad. Sci. 42:203-204.
38. Voglino, P. 1901. Sopra una malattia dei crisantemi coltivati.
Malpighia 15:329.
39. Weiss, F. 1950. Index to plant diseases in the United States.
U. S. Dep. Agr. Plant Disease Survey Special Pub. 1, part 1.
p. 169.


THE DISEASES
The causal organisms of the diseases are Septoria obesa and
Septoria chrysanthemella. Various common names such as leafspot,
black spot, brown spot, septoria leafspot, leaf blight, leaf scorch,
and leaf blotch have been used to describe the diseases. Each of the
fungi has been referred to under most of these common names. Weiss
(39) refers to the disease caused by S. obesa as leaf blotch and to the
disease caused by S. chrysanthemella as leafspot.
Host Range
The disease caused by Septoria obesa was described on Chrysan
themum articum by H. and P. Sydow (36) and later reported on Chrysan
themum morifolium by Hemmi and Nakamura (22). Various investigators
have since found it to be widespread on C. morifolium.
The disease caused by Septoria chrysanthemella was described in
1892 by Cavara (32) on Chrysanthemum morifolium. Since that time it has
been reported on this host by numerous investigators in many parts of
the world. The disease has also been reported on Chrysanthemum
cinerariaefolium (23) and on Chrysanthemum maximum (14). However, cross
inoculation tests have not been made, and, consequently, proof that the
diseases are identical is lacking.
2


3D
TABLE 4
DIMENSIONS AND NUMBER OF SEPTA OF PYCNIDIOSPQHES AND DIAMETERS
OF PYCNIDIA OF SEPTORIA CHRYSANTIIE MELLA SPECIMENS
COLLECTED AT VARIOUS LOCATIONS
IN THE UNITED STATES
Pycnidiospore Distensions
Microns
Pycnidiospore
Septations
Pycnidia Diameters
Microns
Specimen
No.a
Length
Range Meanb
Width
Range
Range
Range
1
25-70
37.7
1.9-2.9
0-5
44-102
2
25-69
40.4
1.8-3.0
0-5
46-124
3
24-50
35.5
2.0-3.0
0-5
40-96
4
22-68
39.8
1.9-2.8
0-5
51-116
Overall
22-70
38.4
1.8-3.0
0-5
40-124
aThe sample size was 34 pycnidiospores or pycnidia.
bA
An analysis of variance gave no significant difference in the
mean length of pycnidiospores from different specimens.


Parasitism of Septoria obesa Syd. and S. chrysanthemella
Sacc. on the Cultivated Chrysanthemum
By
HENRY THOMAS WADDELL
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
August, 1959


52
Both Septoria obesa and Septoria chrysaathemella grow slowly on
potato-dextrose-agar medium. Both cultures are black, raised, rough-
surfaced and compact. S. obesa usually makes more vertical growth, and
S. chrysanthemella spreads more rapidly on the surface of the medium.
The manner of germination of pycnidiospores of Septoria obesa
and Septoria chrysanthemella on water agar differs fresa that of spores
submerged in water. On water agar and on potato-dextrose-agar medium
the spores swell, leaving constrictions at the septa and frequently
between the septa. The first germination hyphae formed usually extend
axially from the basal and apical cells of the spores but lateral hyphae
arise a few hours later from other cells of the spores (Fig. 14).
Pycnidiospores germinating in water, potato-dextrose broth, or synthetic
liquid media do not swell greatly or become conspicuously constricted,
and they germinate mainly by hyphae extending axially from the basal
and apical cells (Fig. 15). Occasionally a pycnidiospore germinating
in water will produce lateral hyphae during the early stages of germina
tion, but this usually does not occur until germination is well advanced
and the spore has become almost indistinguishable from the mycelium.
There is also a difference in the rate of germination of pycnidio
spores in liquids and on solid media. Septoria chrysanthemel1a pycnidio
spores on water agar at 20C. begin to germinate in 7 hours, and there
may be 100 per cent germination at the end of 24 hours. In water,
germinated pycnidiospores of this fungus are rare at the end of 24 hours
and the percentage of germination is usually 50 or less at the end of


60
Experiment 5.Four young plants each of 22 chrysanthemum
varieties were inoculated in two groups of 11 varieties each with
isolate No. 2 of Septoria chrysanthemel1a on March 6 and 9, 1959.
All plants were rooted cuttings with 4 to 6 expanded leaves on each
plant. Disease severity was evaluated 5 weeks later.
Experiment 6.Five plants each of 14 chrysanthemum varieties
were removed from pots and planted with 1-foot spacings in a garden
plot early in April, 1959. On April 15, four plants of each variety
were inoculated with isolate No. 4 of Septoria chrysanthemella, and
the plants were kept wet for 3 days afterwards by means of a sprinkler.
The plants were six weeks old when inoculated. The control group
consisted of 1 plant each of the varieties. Disease severity was
evaluated 5 weeks after inoculation.
RESULTS.Experiment 1.All chrysanthemum varieties artificially
inoculated in the field with Septoria obesa became severely diseased.
Most of the control plants had a few lesions, but these were confined
to leaves near the soil. The data are given in Table 19.
Experiments 2 and 3.Young and old plants in the greenhouse were
severely infected by Septoria obesa, irrespective of the chrysanthemum
variety. Data on these two experiments are summarized in Table 20.
Experiments 4 and 5.Septoria chrysanthemel1a attacked young
and old plants in the greenhouse with equal severity, but there were
definite differences in the pathogenicity of this fungus on various
chrysanthemum varieties. Data are shown in Table 21.
Experiment 6.There were marked differences in disease severity
on different chrysanthemum varieties artificially inoculated in the
field with Septoria chrysanthemella. Data are shown in Table 22.


58
Plants in the greenhouse experiments were inoculated by
atomizing both surfaces of all leaves with spore suspensions made
from pure cultures of the isolates. Immediately after inoculation the
plants were placed in a mist chamber for S days and then removed to a
greenhouse bench. The positions of the plants in the mist chamber and
on the greenhouse beach were always randomized. A control group con
sisting of one plant of each variety used in the particular experiment
was not inoculated with spores of these fungi but otherwise received
the same treatment as did the inoculated plants.
Plants in experiments conducted in outdoor plots were inoculated
with spore suspensions applied with a pressure sprayer. Control plants
were covered by plastic bags during the inoculation operation.
In evaluating the severity of the disease produced by Septoria
chrysanthemella oily the number of lesions formed was considered. Spots
produced by this fungus seldom exceeded one centimeter in diameter. Hie
scale used considered the average number of spots per leaf on the lower
four leaves. Four classes were used in this rating as follows:
Severe: Average of 8 or more spots per leaf.
Moderate: Average of 3 to 7 spots per leaf.
Mild: Average of 1/4 to 2 spots per leaf.
Healthy: Ho infection.
Hie severity of the disease caused by Septoria obesa cannot be
accurately judged by the number of lesions per leaf, as single spots
frequently cover one-quarter or more of a leaf. After observing the
disease produced in the first inoculation experiment the following
scale was formulated which subsequently proved to be applicable in all
other inoculation experiments with S. obesa:


17
TABLE 2
MORPHOLOGICAL CHARACTERS OF SEPTORIA CHRYSANTKEMELLA
AS REPORTED BY DIFFERENT INVESTIGATORS
Morphological
Character
Investigators
Cavara (32)
Rostrup (33)
Hemmi &
Nakamura (22)
Diameter of pycnidia
100-120 u
53-148 u
Length of pycnidiospores
5565 u
40-50 u
23-72 u
Width of pycnidiospores
1.5-2 u
2 u
1.2-3 u
Number of septa
0
0-5
Shape of fehi^Q^^res
* ^
filiform
filiform
filiform
In the summer of 1958 diseased chrysanthemum leaves were obtained
froga various parts of the United States. Of the 16 leaf specimens found
to be infected with Septoria, 12 were infected with Septoria obesa and
4 with Septoria chrysanthemella. Only 8 of the specimens infected with
S. obesa were used in this and other studies in this paper; all 4 S.
chrysanthemgl1a samples were used. The geographical sources, specimen
numbers, and the numbers assigned isolations are given below:
Septoria obesa
Specimen and
Isolation No. Geographical Source
1
2
3
4
5
6
7
8
Gainesville, Florida
Ithaca, New York
Bloomington, Indiana
Urbana, Illinois
Ashtabula, Ohio
Bel Air, Maryland
Spartanburg, South Carolina
Ames, Iowa


3
Geographical Distribution
As previously noted, the two diseases have probably often been
confused. Consequently, reports on geographical distribution may not
be entirely correct.
The disease caused by Septoria obesa was reported by Hemmi and
Nakamura (22) to occur in Japan, Formosa, and the United States. Loos
(24) described the disease in Ceylon in 1941. According to Weiss (39)
it has been reported in Connecticut, Maryland, New Jersey, New York,
Texas, and Washington but is probably much more widespread. In addition
to some of the states listed above by Weiss, the writer has collected
the disease specimens on the leaves of the host in Florida, Indiana,
Illinois, Ohio, South Carolina, North Carolina, Iowa, Missouri, and
Tennessee.
The disease caused by Septoria chrysanthemella has been reported
on Chrysanthemum morifolium in China (22), Japan (22), Formosa (22),
Denmark (33), France (5), the Azores (2), Germany (30), Belgium (26),
Czechoslovakia(28), England (33), Mozambique (8), Iceland (7), Italy
(32), Costa Rica (27), and New Zealand (4). In the IMited States the
disease was noted by Halstead (20, 21) in New Jersey as early as 1891
and was noted by Beach (1) in New York in 1893. According to Weiss
(39) the disease occurs throughout the eastern and central states to
Florida and Texas and also occurs in Colorado and California.
The writer's investigation strongly indicates that the disease
caused by Septoria obesa is much more prevalent in the United States
than is the disease caused by Septoria chrysanthemella. Specimens of
diseased chrysanthemum leaves from 13 southern, eastern, and central


51
Fig, 13.Growth characteristics of Septoria
chrysantheraella on serai-synthetic agar medium sup
plied with different nitrogen sources (1) L-asparagine,
(2) L-arginine, (3) glycine, (3) potassium nitrate,
(5) ammonium sulfate, (6) L-leucine, (7) minus nitrogen.


Parasitism of Septoria obesa Syd. and S. chrysanthemella
Sacc. on the Cultivated Chrysanthemum
By
HENRY THOMAS WADDELL
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
August, 1959

ACKNOWLEDGMENTS
The writer greatly appreciates the guidance, helpful sug
gestions, and aid in manuscript preparation given to him by Dr.
George F. Weber, chairman of his supervisory committee. Appreciation
is expressed to the other members of the supervisory committee
Dr. Mildred Griffith, Dr. Phares Decker, Dr. P. H. Senn, and Prof.
Albert Mullerfor their help and consideration.
Acknowledgments are gratefully made to the University of
Florida Graduate Council, the Southern Fellowships Fund, and the
National Science Foundation for fellowships without which this work
could not have been completed.
Sincere gratitude is expressed to Myra, the devoted wife of
the writer, for encouragement and for the performance of innumerable
tasks, including the typing of this manuscript.
ii
V / (? 51

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS ii
LIST OF TABLES iv
LIST OF FIGURES vi
INTRODUCTION 1
THE DISEASES 2
Host Range ....... ...... 2
Geographical Distribution ... 3
Economic Importance .... 4
Symptoms ..... . 5
THE CAUSAL ORGANISMS 12
Taxonomy 12
Morphology 16
Physiology ....... 36
Pathogenicity ..... 57
HOST-PARASITE RELATIONS 66
Seasonal Development of the Disease 66
Sources of the Inoculum 66
Spore Dissemination 68
Climatic Conditions Affecting Infection and Disease
Development .. 69
Host Penetration 70
Pathological Anatomy ...... 72
SUMMARY 77
LITERATURE CITED 79
BIOGRAPHICAL SKETCH 82
iii

LIST OF TABLES
Table Page
1. Morphological Characters of Septoria obesa as
Reported by Different Investigators . 16
2. Morphological Characters of Septoria chrysanthemella
as Reported by Different Investigators ... 17
3. Dimensions and Number of Septa of Pycnidiospores and
Diameters of Pycnidia of Septoria obesa Specimens
Collected at Various Locations in the United States 29
4. Dimensions and Number of Septa of Pycnidiospores and
Diameters of Pycnidia of Septoria chrysanthemella
Specimens Collected at Various Locations in the United
States ...... ...... 30
5. Comparison of Pycnidiospore Dimensions on Six Chrysan
themum Varieties Inoculated with Isolate No. 1 of
Septoria obesa 31
6.Comparison of Pycnidiospore Dimensions on Six Chrysan
themum Varieties Inoculated with Isolate No. 1 of
Septoria chrysanthemella ....... 32
7. Comparison of Septoria obesa and Septoria chrysanthe
mella Specimens Collected in the United states:
Pycnidiospores and Pycnidia 34
8. The Effect of Hydrogen-ion Concentration on the Growth
of Septoria obesa in a Synthetic Liquid Medium ... 40
9.The Effect of Hydrogen-ion Concentration on the Growth
of Septoria chrysanthemel1a in a Synthetic Liquid
Medium 41
10.The Effect of Temperature on the Growth of Septoria
obesa in Synthetic Liquid Media 42
11.The Effect of Temperature on the Growth of Septoria
obesa on Semi-synthetic Agar Medium 43
12.The Effect of Temperature on the Growth of Septoria
chrysanthemella in Synthetic Liquid Meditan ..... 44
iv

LIST OF TABLES (continued)
Table Page
13.The Effect of Temperature on the Growth of Septoria
chrysanthemella on Semi-synthetic Agar Medium .... 45
14.The Effect of Carbon Source on the Growth of
Septoria obesa on Semi-synthetic Agar Medium .... 47
15.The Effect of Carbon Source on the Growth of
Septoria chrysanthemella on Semi-synthetic Agar Medium 48
16,The Effect of Nitrogen Source on the Growth of
Septoria obesa on Semi-synthetic Agar Medium .... 49
17. The Effect of Nitrogen Source on the Growth of
Septoria chrysanthemella on Semi-synthetic Agar
Medium ........ 50
18. Percentage of Germination of Pycnidiospores on Water
Agar at Various Temperatures ..... 54
19. Severity of Disease on Mature Plants of Fifteen
Chrysanthemum Varieties Inoculated with Septoria obesa
in the Field 61
20. Severity of Disease on Twenty-two Chrysanthemum
Varieties Inoculated with Septoria obesa in the Green
house 62
21. Severity of Disease on Twenty-two Chrysanthemum
Varieties Inoculated with Septoria chrysanthemella
in the Greenhouse ............ 63
22. Severity of Disease on Fourteen Chrysanthemum
Varieties Inoculated with Septoria chrysanthemella
in the Field 64
v

LIST OF FIGURES
Figure Page
1.Lesions on Old Chrysanthemum Leaves 6 Weeks after
Inoculation with Spores of Septoria obesa .. 8
2.Lesions on Young Chrysanthemum Leaves 6 Weeks after
Inoculation with Spores of Septoria obesa 9
3.Lesions on Old Chrysanthemum Leaves 6 Weeks after
Inoculation with Spores of Septoria chrysanthemella ... 10
4,Lesions on Young Chrysanthemum Leaves 8 Weeks after
Inoculation with Spores of Septoria chrysanthemella . 11
5. Longitudinal Section of a Pycnidium of Septoria obesa
in Chrysanthemum Leaf Tissues ...... 21
6. Pycnidiospores of Septoria obesa 22
7. Pycnidia of Septoria obesa on Potato-Dextrose-Agar
Medium . . . . 23
8. Longitudinal Section of a Pycnidium of Septoria chrysan-
themella in Chrysanthemum Leaf Tissues ......... 25
9. Pycnidiospores of Septoria chrysanthemella ....... 26
10. Pycnidia of Septoria chrysanthemella on Potato-Dextrose-
Agar Medium ........... 27
11. Camera Lucida Drawing of Pycnidiospores of Septoria
chrysanthemella and Septoria obesa ........... 35
12. Growth of Septoria obesa and Septoria chrysanthemella
on Semi-synthetic Agar Medium at Optimum Temperatures
at the End of 40 Days 46
13. Growth Characteristics of Septoria chrysanthemella on
Semi-synthetic Agar Medium Supplied with Different Nitrogen
Sources (1) L-asparagine, (2) L-arginine, (3) glycine (4)
potassium nitrate, (5) ammonium sulfate, (6) L-leucine,
(7) minus nitrogen ............. 51
vi

LIST OF FIGURES (continued)
Figure Page
14, Pycnidiospores of Septoria chrysanthemel1a Germinat
ing on Solid Medium .......... 53
15, Pycnidiospores of Septoria chrysanthemella Germinat
ing in Water 53
16, Stomatal Penetration by a Hypha from a Germinating
Pycni dio spore of Septoria obesa 73
17. Stomatal Penetration by a Hypha from a Germinating
Pycnidiospore of Septoria chrysanthemella 73
13. Cross-section of Host Leaf Shoving Intercellular
Invasion of Mesophyll Tissue by Septoria obesa
Mycelium ... 75
19. Cross-section through a Leaf Lesion Showing Pycnidia
of Septoria obesa 76
20. Cross-section through a Leaf Lesion Shoving Pycnidia
of Septoria chrysanthemella 76
vil

INTRODUCTION
Two distinct septoria diseases of the cultivated chrysanthemum
commonly occur in the United States and in several other parts of the
world where the host is grown.
The development of similar disease symptoms has led to un
certainty and error. Confusion abounds with respect to the identity
of the causal organism. Information is incomplete or lacking cm the
morphology and physiology of the causal parasites and also on their
relative pathogenicity on various varieties of the cultivated chrysan
themum. Various host-parasite relationships are not known, especially
the mode of penetration by the fungus and pathological anatomy.
These investigations were inaugurated to explore those areas
where information is incomplete or lacking with respect to the
diseases, the pathogens, and the host-pathogen relations.
The results are reported herewith. The experimental work is
supplemented by a review of the literature in all areas pertaining
to the diseases, the pathogens, and the host-pathogen relations.
1

THE DISEASES
The causal organisms of the diseases are Septoria obesa and
Septoria chrysanthemella. Various common names such as leafspot,
black spot, brown spot, septoria leafspot, leaf blight, leaf scorch,
and leaf blotch have been used to describe the diseases. Each of the
fungi has been referred to under most of these common names. Weiss
(39) refers to the disease caused by S. obesa as leaf blotch and to the
disease caused by S. chrysanthemella as leafspot.
Host Range
The disease caused by Septoria obesa was described on Chrysan
themum articum by H. and P. Sydow (36) and later reported on Chrysan
themum morifolium by Hemmi and Nakamura (22). Various investigators
have since found it to be widespread on C. morifolium.
The disease caused by Septoria chrysanthemella was described in
1892 by Cavara (32) on Chrysanthemum morifolium. Since that time it has
been reported on this host by numerous investigators in many parts of
the world. The disease has also been reported on Chrysanthemum
cinerariaefolium (23) and on Chrysanthemum maximum (14). However, cross
inoculation tests have not been made, and, consequently, proof that the
diseases are identical is lacking.
2

3
Geographical Distribution
As previously noted, the two diseases have probably often been
confused. Consequently, reports on geographical distribution may not
be entirely correct.
The disease caused by Septoria obesa was reported by Hemmi and
Nakamura (22) to occur in Japan, Formosa, and the United States. Loos
(24) described the disease in Ceylon in 1941. According to Weiss (39)
it has been reported in Connecticut, Maryland, New Jersey, New York,
Texas, and Washington but is probably much more widespread. In addition
to some of the states listed above by Weiss, the writer has collected
the disease specimens on the leaves of the host in Florida, Indiana,
Illinois, Ohio, South Carolina, North Carolina, Iowa, Missouri, and
Tennessee.
The disease caused by Septoria chrysanthemella has been reported
on Chrysanthemum morifolium in China (22), Japan (22), Formosa (22),
Denmark (33), France (5), the Azores (2), Germany (30), Belgium (26),
Czechoslovakia(28), England (33), Mozambique (8), Iceland (7), Italy
(32), Costa Rica (27), and New Zealand (4). In the IMited States the
disease was noted by Halstead (20, 21) in New Jersey as early as 1891
and was noted by Beach (1) in New York in 1893. According to Weiss
(39) the disease occurs throughout the eastern and central states to
Florida and Texas and also occurs in Colorado and California.
The writer's investigation strongly indicates that the disease
caused by Septoria obesa is much more prevalent in the United States
than is the disease caused by Septoria chrysanthemella. Specimens of
diseased chrysanthemum leaves from 13 southern, eastern, and central

4
states were examined. The Septoria obesa disease was found on collec
tions from 13 of these states, whereas the Septoria chrysanthemella
disease was found on specimens only from Iowa. Texas, and Tennessee.
Economic Importance
The chrysanthemum is one of the most important flowers of
commerce in the United States and also in several other parts of the
world. Most of the plants are ultimately sold as cut flowers, but there
is also a large trade in potted plants and in rooted cuttings for plant
ing in private gardens.
The septoria leafspot diseases were very severe in commercial
beds in the United States until World War II. The diseases are now
controlled to a great extent in commercial plantings by dithiocarbamate
sprays but frequently become severe during extended rainy periods. The
diseases remain a problem in private gardens where control measures are
usually less rigidly practiced.
The writer agrees with the statements of Dimock (10) concerning
losses due to septoria leafspots:
1. Spotting of foliage seriously detracts from the
quality and salability of cut flowers.
2. Even if spotting is confined to the lower leaves,
the loss of lower foliage may seriously reduce both
the quantity and quality of blooms.
As previously stated, the disease caused by Septoria obesa is
apparently more common than the Septoria chrysanthemella disease in the
United States, and. as will be described later, it is considerably more
severe,

5
Symptoms
The following descriptions are based on observations of the
natural development of the diseases in the field and greenhouse and on
inoculation experiments under various controlled conditions*
DESCRIPTIONS.The disease caused by Septoria obesa is usually
considerably more severe than the disease caused by Septoria chrysanthe-
mella. Lesions begin as small chlorotic areas which usually become
dark brown or black and enlarge to form circular to very irregular areas
with distinct margins* Frequently* however* the young lesion develops
into a blackish or brownish blotch-like lesion of indefinite size and
shape with indistinct margin. The latter type of lesion bocones black
and the margin becomes distinct during rainy weather* A yellow halo
usually surrounds the lesion* A single lesion may spread to occupy
1/3 or more of a leaf* and a leaf may be killed by one or two lesions*
The lesions are distinct on both surfaces of the leaf but are darker
cm the adaxial surface. Pycnidia are usually abundant cm both surfaces
of the lesions*
The disease attacks and kills the lower leaves first and under
favorable conditions progresses upward* finally attacking the upper
leaves and the lnvolucral bracts* Entire shoots or entire plants are
frequently killed* The symptoms are shown in Figs* 1 and 2*
The disease caused by Sep tori a chrysanthemell a occurs cm
susceptible chrysanthemum plants of all ages* The first symptom is the
appearance of very small chlorotic spots* barely discernible without
magnificat!cm* cm the leaf blades* Under favorable moisture and humidity

6
conditions, these spots bee cm black in a few days and gradually en
large to form black, circular or elliptical spots, mostly 1 cm or less
in diameter but sometimes as large as 2 cm in diameter. These lesions
usually show distinct margins, and they are sometimes surrounded by
narrow chlorotic halos. They are visible on both surfaces of the leaf
but are considerably more conspicuous on the adaxial surface.
When the adaxial surface of a lesion is examined under a hand
lens, numerous black pycnidia are seen embedded in the leaf tissue.
There may be only a few lesions on an infected leaf, or there may be
so many that they coalesce, thus forming large necrotic areas. The
centers of old lesions sometimes become grayish in color.
The lower leaves of a plant are infected first, and if infection
is severe they die and hang downward on the stem. Following periods of
rainfall, the disease progresses upward on the plant, and the uppermost
leaves and the involucra! bracts may eventually become infected.
Symptoms of this disease are shown in Figs. 3 and 4.
COMPARISON.Advanced symptoms produced by Septoria obesa under
favorable conditions of moisture and temperature are distinctly dif
ferent from those produced by Septoria chrysanthemella, the leaf lesions
being large and circular to irregular in shape. S>. chrysanthemella
causes circular to elliptical leaf lesions which usually do not exceed
1 cm in diameter, but these lesions may coalesce to form black, irregular
shaped, necrotic areas; however, when closely observed, the individual
spots making up the irregular areas are easily discerned. Another point
of difference is the occurrence of abundant pycnidia chi the abaxial
surface of S. obesa lesions, whereas S. chrysanthemella lesions either

7
lack or have only a few pycnidia on their ahaxial surfaces*
Young lesions produced by Septoria obesa are frequently small
and circular, thus resembling those of Septoria chrysanthemella* The
two diseases can be distinguished with certainty by observing the
pycnldiospores produced in the lesions. The morphology of the pycnidio-
spores will be described later.

3
Fig. 1,Lesions on old chrysanthemum leaves
6 weeks after inoculation with spores of Septoria
obesa (X 1/3).

9
Fig. 2.Lesions on young chrysanthemum leaves 6 weeks
after inoculation with spores of Septoria obesa (X 3/4).

Fig. 3.--Lesions on old chrysanthemum leaves
6 weeks after inoculation with spores of Septoria
chrysanthemella (X 1/3).

Fig. 4.--Lesions on y e're.yr.aathemuin leaves
8 weeks after inoculation with spores of Septoria
chrvsanthemel1a (X 3/4).

THE CAUSAL ORGANISMS
Taxonomy
The identity of the numerous species of Septoria is based on
morphological differences, physiological adaptations, and specificity
for different host plants. Regardless of morphological similarities,
Septoria spp. specific for different, not-closely-related hosts are
placed in separate species. Morphologically similar septort sis which
are specific for different but closely-related hosts, such as hosts
in the same genus or family, are usually placed in different species
but are sometimes given varietal names in the same species.
In discussing the taxonomy of the two causal organisms under
consideration, septorlas described on different host plants will be
considered to be of different species unless proved identical by
cross-inoculation experiments.
SEPTORIA CHRYSANTHEMELLA SACC.In 1892 Cavara (32) described
a fungus causing spots on chrysanthemum leaves in Italy. He gave
this fungus the binomial Septoria chrysanthemi Cav. However, since
Allescher (32) had used this name in 1891 in describing a very similar
fungus on the leaves of Chrysanthemum leucanthemum in Bavaria, Saccardo
(32), in 1895, changed the name of Cavara*s fungus to Septoria chrysan-
themella Cav. In 1907 Magnus (25) stated that the latter fungus should
12

13
be referred to under the name of Septoria chrysanthemella Sacc.t as
the name had been changed by Saccardo and not by Cavara.
In 1897 Rostrup (33) in Denmark, described a fungus parasitic
on the chrysanthemum under the name of Septoria chrysanthemi Rostr.
Due to the presence of Alleschers species, Saccardo and Sydow (33)
changed the name to Septoria rostrupii Sacc. and Syd. in 1899. Magnus
(25) and Hemmi and Nakamura (22) state that this fungus is the same
as Septoria chrysanthemella Sacc. After studying the descriptions of
the two fungi, this writer agrees with them.
In 1907 in Bohemia, Bubak and Kabat (34) described a fungus
attacking the chrysanthemum under the name of Septoria chrysanthemi-
indici Bub. & Kab. Magnus (25) believed this fungus to be synonymous
with Septoria chrysanthemella Sacc. If the original description is
accurate the opinion of Magnus is incorrect, for the pycnidiospores
of S. chrysanthemella are filiform and 0-5 septate, whereas Bubak and
Kabat described the pycnidiospores of their fungus as being tapered
from the base to the apex and 10-15 septate. According to this descrip
tion the pycnidiospores of the latter fungus are similar to those of
Septoria obesa Syd. When the type specimen of Bubak and Kabat was
examined by Hemmi (22), he found that the pycnidiospores were not as
described, being more similar to those of S. chrysanthemella Sacc.
Since more than one species of Septoria may occur on the same plant,
the synonymy of S. chrysanthemi-indicl with either S. chrysanthemella
Sacc. or S. obesa Syd. is questionable.
According to Chifflot (5) Septoria variaos Joffrin is a synonym
of Septoria chrysanthemella. The writer has not had an opportunity

14
to read Joffrin's description of this fungus, but as described by
Chifflot the pycnidiospores are filiform and many-celled, and figures
of the spores shoe than to be obclavate and 10-14 septate* If
Chifflot's description is accurate, Septoria varians is not the same
as Septoria chrysanthemella.
The valid name and synonyms of the fungus under consideration
are as follows:
Septoria chrysanthemella Sacc. (sub nom. Septoria
chrysanthemella Cav* 1895)
Synonyms:
Septoria chrysanthemi Cav* 1892*
Septoria chrysanthemi Rostr* 1897*
Septoria rostrupii Sacc. & Syd* 1899*
SEPTORIA OBESA SYD.*In 1914 H. and P* Sydow (36) described a
new fungus, Septoria Obesa Syd., parasitic on Chrysanthemum arcticum,
a wild plant of northern Japan.
In 1917 Hemmi (22) found too different septorias parasitic on
Chrysanthemum morifolium in Japan. One species was identified as
Septoria chrysanthemella. The other species was first identified as
Septoria chrysantheml-indici Bub. & Kab., but after examining the type
specimen of this fungus, Hemmi reversed his opinion. Subsequently
Hemmi studied Sydow*s description of Septoria obesa and examined the
type specimen. He found that his species was morphologically identical
to Septoria obesa Syd. and stated that the two are synonymous. Theo
retically, Hemmi's species should have been described as a new species
or as a variety of S. obesa. The two occurred on different hosts and
cross-inoculation experiments were not conducted.

In 1893 Ellis and Dearness (15) in Canada described a fungus
parasitic on chrysanthemums and named it Cyllndrosporlua chrysantheml
15
E. ft D. The spores of this fungus were very similar to those of
Septoria obesa Syd. and the spore-bearing structures were similar in
shape and size to pycnidia of S. obesa Sydbut lacked a pycnidial vail.
The spore-bearing organ vas therefore considered to be an acervulus
and not a pycnidlum. Trent (37) suggests that this fungus is synonymous
with Septoria chrysanthemella Sacc., and Greene (18) states that it may
be the same as Septoria obesa Syd. Unless the original description can be
shown to be wrong, the writer cannot consider this fungus to be classi
fied in the genus Septoria.
Weiss (39) lists Septoria leucanthemi Sacc. ft Spec., Septoria
macrosporia Dearn., and Septoria cercosporoides Trail as probable
synonyms of Septoria obesa Sacc. The writer cannot agree that any one
?
Of these three fungi is synonymous with S. obesa Sacc., as all three
were described on Chrysanthemum leucanthemum and cross-inoculation
experiments have not been conducted. Furthermore, the pycnidiospores
of both Septoria leucanthemum (31) and Septoria macrosporia (18) are
filiform. This differs from the pycnidiospores of Septoria obesa which
are obclavate. The pycnidiospores of Septoria cercosporoides as given
by Cooke (6) after Trail appear to be very similar to those of S. obesa
but are somewhat smaller.
The valid name of the fungus under consideration is:
Septoria obesa Syd. 1914.
Synonyms: none.

16
Morphology
Both Septoria obesa and Septoria chrysanthemella produce
nycelia, pycnidia, and pycnidiospores in leaf tissues of susceptible
hosts and in suitable artificial culture media. A perfect stage of
either of these fungi has not been reported. Voglino (38) in 1901
reported what he termed a phoma stage of S. chrysanthemella on the
leaves and stems of chrysanthemums. This stage has not been observed
by any other investigator, and it seems likely that it was produced
by a fungus distinct from S. chrysanthemella.
Several authors have given descriptions of the pycnidia and
pycnidiospores of Septoria obesa and Septoria chrysanthemella. A
summary of these descriptions is given in Tables 1 and 2.
TABLE! 1
MORPHOLOGICAL CHARACTERS OF SEPTORIA OBESA AS REPORTED
BY DIFFERENT INVESTIGATORS
Investigators
Morphological
Character
H. & P.
Sydow (36)
Hemrni &
Nakamura (22)
Loos (24)
Diameter of pycnidia
120-160 u
75-200 u
87-173 u
Length of pycnidiospores
50-100, rarely 41-124 u
120 u
66-87 u
Width of pycnidiospores
3-4.5 u
2.5-4.4 u
3.2-3.6 u
Number of septa
5-12
4-12
up to 9
Shape of pycnidiospores
narrowly
obclavate
whip-shaped
Rounded at
base, tapered
toward apex

17
TABLE 2
MORPHOLOGICAL CHARACTERS OF SEPTORIA CHRYSANTKEMELLA
AS REPORTED BY DIFFERENT INVESTIGATORS
Morphological
Character
Investigators
Cavara (32)
Rostrup (33)
Hemmi &
Nakamura (22)
Diameter of pycnidia
100-120 u
53-148 u
Length of pycnidiospores
5565 u
40-50 u
23-72 u
Width of pycnidiospores
1.5-2 u
2 u
1.2-3 u
Number of septa
0
0-5
Shape of fehi^Q^^res
* ^
filiform
filiform
filiform
In the summer of 1958 diseased chrysanthemum leaves were obtained
froga various parts of the United States. Of the 16 leaf specimens found
to be infected with Septoria, 12 were infected with Septoria obesa and
4 with Septoria chrysanthemella. Only 8 of the specimens infected with
S. obesa were used in this and other studies in this paper; all 4 S.
chrysanthemgl1a samples were used. The geographical sources, specimen
numbers, and the numbers assigned isolations are given below:
Septoria obesa
Specimen and
Isolation No. Geographical Source
1
2
3
4
5
6
7
8
Gainesville, Florida
Ithaca, New York
Bloomington, Indiana
Urbana, Illinois
Ashtabula, Ohio
Bel Air, Maryland
Spartanburg, South Carolina
Ames, Iowa

18
Septoria chrysanthomella
Specimen and
Isolation No.
Geographical Source
1
2
3
4
Troy, Tennessee
Beauiaont, Stexas
Ames, Iowa*
Ames, Iowa*
Specimens from Ames came from different nurseries.
Studies were made of the morphological characters of the fungi
in the original leaf samples, in pure culture, and in the leaves of
several chrysanthemum varieties artificially inoculated with pure
cultures of the pathogens.
METHODS AND MATERIALS.In the observations of pycnidiospores
in the original leaf specimens, randomization of the spores was obtained
by soaking several lesions of each specimen in water for three hours and
then stirring the resulting spore suspensions vigorously. In order to
stain the septa of the pycnidiospores, an iodine solution, was added to
the suspensions. Drops of the suspensions were then placed on glass
slides and pycnidiospores measured by use of a previously calibrated
ocular micrometer. Septoria obesa pycnidiospores were measured for
width near their bases, whereas those of Septoria chrysaathemella were
measured for width midway between their bases and apices. Lengths and
widths were measured at 440 and 970 magnifications, respectively. A
mechanical stage was used to insure that no spore was measured more
than once.
The procedure used in securing isolations from the original
leaf specimens was as follows: Hie diseased leaves were soaked in

19
1:1000 mercuric chloride for 2 minutes and then for 5 minutes each in
3 changes of sterile distilled water. The diseased leaves were then
placed on moist filter paper in sterile petri dishes and maintained
for 24 hours in a constant temperature chamber at 8C. At the end of
this period pycnidiospores in gelatinous matrices had oozed from many
of the pycnidia. With the aid of a stereoscopic microscope, spore
suspensions were prepared by touching the spore masses with the tip
of a sterile needle and rinsing the needle tip in sterile, distilled
water. The suspensions were then plated on water agar in petri dishes
and incubated for about 43 hours at 20C. Bacterial contaminants had
formed colonies by the end of this period and were easily avoided.
Germinated spores were removed singly with a hooked needle and trans
ferred to potato-dextrose-agar medium in petri dishes.
Measurements of pycnidiospores obtained from several chrysan
themum varieties previously artificially inoculated with isolate No. 1
of Septoria chrysanthemella or with isolate No. 1 of Septoria obesa
were made by using the procedures described earlier in this section.
The artificially inoculated material was from an experiment concerned
primarily with pathogenicity, and an account of the techniques employed
is given under that topic.
Studies of morphological characters of the fungi in host leaves
and in culture on potato-dextrose-agar medium were made from serial
sections prepared by the paraffin method and examined unstained or
after being stained with safranin, safranin-fast green, cotton blue,
or iron hematoxylin

20
RESULTS.The following descriptions of the morphological
characters of the two species of Septoria under consideration are
based on the studies described above.
Septoria obesa.Pycnidia formed in host tissues are typically
globose or subglobose but sometimes pyriform or irregular in shape,
60-160 microns in diameter. Short to rather elongate beaks are present,
and the diameters of ostioles vary from almost no opening to that
measuring one-half the diameter of the pycnidium. A pycnidium of S.
obesa is shown in the leaf tissues of the chrysanthemum in Fig. 5.
Pycnidial walls are composed of 3 to 7 layers of cells which are
isodiametric to elongate in shape. The inner layer of cells is hyaline,
and the outer layers are brown. Conidiophores are composed of cells
of the inner wall of the pycnidium which have became slightly elongated;
they are hyaline, 3-7 microns in length. The mycelium is mostly 3
microns or less but sometimes up to 6 microns in diameter, finely
granular, hyaline, constricted at the septa, obtusely branched, and
septate at the points of branching. A mature pycnidiospore of S. obesa
is relatively large and rounded at the base and tapers from the basal
end or from near the mid-region of the spore to the apex (Fig. 6). In
culture on potato-dextrose-agar medium the morphology of pycnidiospores
is similar to that described above. Pycnidia vary greatly in shape,
often forming extensive flattened cavities, and the ostioles are not
well defined (Fig. 7). Most of the mycelium is devoted to producing
spherical or elliptical-shaped cells which go into the formation of
pycnidial walls

21
Fig, 5.Longitudinal section of a pycnidium of
Septoria obesa in chrysanthemum leaf tissues (X 476).

22
Fig. 6.Pycaidiospores of Sep tori a obesa (X 47G)

23

24
Septoria chrysanthemella.Pycnidia formed in host tissues
(Fig. g) are mostly pyriform but frequently globose or subglobose in
shape, 40-124 microns in diameter, and slightly to 1/2 erumpent.
Ostioles occur at the ends of short to rather elongate beaks and vary
in diameter from being almost closed to being about 1/2 or more the
diameter of the pycnidiurn when spores are being exuded. Hie walls of
the pycnidia are composed of 3 to 6 layers of compact cells which are
usually nearly isodiametric but sometimes 3 or 4 times as long as broad.
%
Hie outer cell layers are brown in color but the innermost layer is
hyaline. Conidiophores are composed of the inner cells of the pycnidial
wall which have elongated slightly toward the center of the pycnidium.
They are hyaline and 3 to 6 microns in length. Pycnidiospores (Fig. t)>
are filiform, usually straight but sometimes slightly curved, uniform
in width and bluntly pointed at base and apex, obscurely 0-5 septate,
22-70 x 1.8-3 microns, hyaline, finely granular, and usually several
guttulate. The mycelium is similar to that described above for Septoria
obesa.
In culture on potato-dextrose-agar medium, the morphology of
the pycnidiospores of Septoria chrysanthemella is as described above.
Pycnidia in culture are globose to very irregular in shape and the walls
are usually much thicker than in host tissue. The first pycnidia formed
are almost superficial on the surface of the culture and are oriented
with their ostioles communicating with the internal portion of the
culture (Fig. 10). Only occasionally do ostioles open on the surface.
Pycnidia formed in deeper layers of the culture are oriented variously.
Hie morphology of the mycelium is similar to that described for Septoria
obesa,

Fig. 8;Longitudinal section of a pycnidium
of Septoria chrysantheaella in chrysanthemum leaf
tissues (X476).

26
Pig. 9r-Pycnidiospores of Septoria chrysanthe-
aella (X 476).

'7

28
Summaries o pycnidial diameters and pycnidiospore dimensions
and number of septa observed in leaf specimens collected in various
locations in the United States are given in Tables 3 and 4 for Septoria
obesa and Septoria chrysanthemella,respectively. Similar information
is given in Tables 5 and 6 for pycnidiospores produced in different
*
chrysanthemum varieties after artificial inoculations with isolate No. 1
of S. obesa or isolate No. 1 of S. chrysanthemella, respectively.

TABUS 3
DIMENSIONS AND NUMBER OF SEPTA OF PYCNIDIOSPORES AND DIAMETERS OF
FYCNIDIA OF SEPTORIA OBESA SPECIMENS COLLECTED AT
VARIOUS LOCATIONS IN TEE UNITED STATES
Pycnidiospore Dimensions
Microns
Pycnidiospore
Septations
Pycnidia Diameter!
Microns
Specimen
No.a
Length
Range Meanb
Width
Range
Range
Range
1
54-106
78.0
2.8-4.5
7-13
80-156
2
46-104
75.5
2.6-4.0
7-14
74-148
3
45-100
73.2
2.8-4.5
6-13
62-156
4
44-102
72.9
2.7-4.4
6-14
60-123
5
44-100
71.4
2.8-4.4
5-14
75-132
6
50-108
74.7
2.7-4.5
6-14
68-160
7
45-98
74.2
2.6-3.9
5-13
72-149
8
45-100
73.8
2.7-4.3
7-14
70-140
Overall
44-108
74.2
2.6-4.5
5-14
60-160
aThe sample size was 34 pycnidiospores or pycnidia.
An analysis of variance gave no significant difference in the
mean length of pycnidiospores from different specimens.

3D
TABLE 4
DIMENSIONS AND NUMBER OF SEPTA OF PYCNIDIOSPQHES AND DIAMETERS
OF PYCNIDIA OF SEPTORIA CHRYSANTIIE MELLA SPECIMENS
COLLECTED AT VARIOUS LOCATIONS
IN THE UNITED STATES
Pycnidiospore Distensions
Microns
Pycnidiospore
Septations
Pycnidia Diameters
Microns
Specimen
No.a
Length
Range Meanb
Width
Range
Range
Range
1
25-70
37.7
1.9-2.9
0-5
44-102
2
25-69
40.4
1.8-3.0
0-5
46-124
3
24-50
35.5
2.0-3.0
0-5
40-96
4
22-68
39.8
1.9-2.8
0-5
51-116
Overall
22-70
38.4
1.8-3.0
0-5
40-124
aThe sample size was 34 pycnidiospores or pycnidia.
bA
An analysis of variance gave no significant difference in the
mean length of pycnidiospores from different specimens.

31
TABUS 5
COMPARISON OP PYCNIDIOSPORE DIMENSIONS ON SIX CHRYSANTHEMUM
VARIETIES INOCULATED WITH ISOLAIS HO. 1 OP SBPTORIA OBESA
Chrysanthemum Variety
Length in
Microns
Range
Mean*5
Alaska
49-101
74.5
Gold Ball
48-100
74.1
Illini Regal
46-96
71.7
mini Warpaint
53-102
76.0
Jetfire
50-114
74.0
Mary L. Hall
47-99
72.8
Overall
46-114
73.8
aThirty-four pycnidiospores were measured in each variety.
bThere vas no significant difference in mean length of spores
among varieties at the 5 per cent level*

TABLE 6
COMPARISON OF PYCNIDIOS PORE DIMENSIONS ON SIX CHRYSANTHEMUM VARIETIES
INOCULATED WITH ISOLATE NO. 1 OF SEPTORIA CHRYSANTHEMELLA
Chrysanthemum Variety^
Range
Length in Microns
Mean11
Alaska
30-68
38.4
Bonnie
29-72
40.8
Delaware
28-72
39.7
Mary L. Hall
32-74
41.5
Rayonnante
28-58
37.7
Yellow Shasta
25-68
37.2
Overall
25-74
38.9
aThirty-four pycnidiospores were measured in each variety,
b
There was no significant difference in mean length of spores
among varieties at the 5 per cent level.

DISCUSSION.Cavara (32) described the pycnidiospores of
Septoria chrysanthemella as being non-septate; however, the septa in
this species are usually invisible unless the spore walls are artificially
stained. Hemal and Nakamura (22) describe the pycnidiospores of Septoria
obesa as being obscurely septate, and these investigators used an iodine
stain in order to observe the septa. The pycnidiospores of S. obesa
observed by the writer were all distinctly septate if well matured. It
is possible that Hemmi and Nakamura observed the spores just prior to
their full maturity, or there may be a difference in the distinctness of
septations in the species in different parts of the world or under dif
ferent environmental conditions.
The two species of Sentoria on the chrysanthemum collected in
the United States are Septoria chrysaathemella and Septoria obesa.
There was no significant difference in the mean length of either
Septoria obesa pycnidiospores or Septoria chrysaathemella pycnidiospores
produced on six different chrysanthemum varieties. It cannot be stated
that the host variety does not affect this character, as a comparatively
small number of varieties was used, and they were not selected at random
from the several thousand varieties in cultivation.
No significant difference was found in the mean length of either
Septoria obesa or Septoria chrysanthemella pycnidiospores collected from
different locations in the United States. Geographical locations were
not randomly selected. Pycnidiospore length could possibly be affected
by both environmental conditions and hereditary factors.
There was a considerable difference between the dimensions and
number of septa of pycnidiospores and also between the pycnidial

34
dimensions o the two Septoria species. The mean length of the
pycnidiospores of Septoria chrysaathemella was about one-half that of
the mean length of those of Septoria obesa, and the maximum number of
septa in the pycnidiospores of the former species was the same as the
minimum in the pycnidiospores of the latter species. The pycnidia of
S. obesa were usually larger than those of S. chrysanthemella and also
differed markedly in shape. A comparison of the pycnidiospores and
pycnidia of the two Septoria species is shown in Table 7.and Fig. 11.
TABLE 7
COMPARISON OF SEPTORIA OBESA AND SEPTORIA CHRYSANTHEMELLA SPECIMENS
COLLECTED IN THE UNITED STATES: PYCNIDIOSPORES AND PYCNIDIA
Morphological Character
Septoria obesa
S. chrysanthemella
Pycnidiospores:
Range in length
44-108 u
22-70 u
Mean length
74.2 u
38.4 u
Range in width
2.6-4.5 u
1.8-3.0 u
Mean width
3.2 u
2.1 u
No. of septa
5-14
0-5
Shape
Tapered from base
or mid-region to
apex. Usually
curved.
Linear. Usually
straight or slightly
curved.
Pycnidia:
Range in diameter
60-160 u
40-124 u
Mean diameter
92 u
68 u

Fig. 11.--Camera lucida drawing of pycnidio-
spores of Septoria chrysanthemella (left)and
Septoria obesa (right).

36
Physiology
In 1927 Herami and Nakamura (22) investigated the effect of
temperature cm the growth of Septoria chrysanthemella and Septoria
obesa on solid media. The results of this investigation will be dis
cussed later in this section. The same investigators also observed the
growth of the two fungi in various solid and liquid media, but these
experiments were not designed to give information on the specific
nutritive requirements of the two fungi.
The effects on the growth of the fungi in cultures of different
carbon sources, nitrogen sources, pH levels, and temperature levels are
reported herewith. Studies were made of pycnldiospore germination, and
the habits of growth of the two fungi on potato-dextrose-agar medium
were compared.
METHODS AND MATERIALS. The growth of the fungi was observed on
potato-dextrose-agar medium; water was used as a medium in spore-
germination studies. The culture medium employed otherwise was a modi
fication of a basal synthetic medium described by Hacskaylo et al.
(19). The formula for the preparation of one liter of this medium and
the compounds used unless otherwise stated are as follows:
Carbon source
N
kh2po4
MgS047H20
Fe444
Zn444
Mn44
10.0 g (dextrose)
425 mg (2.000 g, L-asparagine)
1.0 g
0.50 g
0.2 mg Cl.006 mg, Fe2 (804)3*9^0}
0.2 mg (.8796 mg, ZnS047H20)
0.1 mg (.4077 mg, ¡OaSO^^O)
Biotin 5 ug
Thiamine 100 ug
H20 (Distilled) to make 1 liter of solution.
Twenty grams of agar per liter of solution were used when solid
media were prepared.

37
Stock solutions of the iron zinc manganese and vitamin
sources were prepared at concentrations 1000 times those used in the
culture media.
The medium described above will hereafter be referred to as
synthetic liquid medium or if agar is added as semi-synthetic agar
medium.
In all experiments in which synthetic liquid medium was used
each replication of the medium was inoculated with the fungus by adding
5 drops of a heavy suspension of germinated pycnidiospores and each
culture was agitated once a day by shaking. The spore suspensions
were prepared by flooding one-month-old potato-dextrose-agar sporu-
lating cultures of the fungi with sterile distilled water. A dif
ferent isolate was used in each replication of the experiments con
cerned with pH, temperature, carbon source, and nitrogen source.
Filter papers were weighed individually after drying in an oven for
2 days at 44C. The mycelium was collected on these weighed filter
papers with the aid of a Buchner funnel under vacuum. The papers and
collected mycelium were weighed after drying and the differences com
puted. All weights of mycelium were determined to the nearest 0.1 mg.
The effect of hydrogen-ion concentration on the growth of the
two fungi in synthetic liquid medium was investigated at pH units of
2 through 10. The pH of the medium was adjusted after autoclaving by
adding measured amounts of 0.1N, IN, and 5N HC1 or NaOH to 125 ml
portions of the medium in 250 ml erlenmeyer flasks. The amounts of
acid or base necessary to give the desired pH level were previously
determined with the aid of a Beckman pH meter. Each replication was

inoculated with 3-day old germinating pycnidiospores and incubated for
28 days at 20C. Dry mycelial weights were then determined. Four
replications were used for Septoria chrysanthemella and 6 for Septoria
38
obesa. The experiments were not conducted concurrently for the two
species of Septoria, but all conditions were duplicated as nearly as
possible.
The effects of different temperatures on the growth of the fungi
in both synthetic liquid medium and semi-synthetic agar medium were
investigated. Germinating spores in 125 ml portions of the liquid
medium were incubated for 28 days at four-degree intervals from 4 to
32C., and mycelial weights were then determined. Three replications
were used for Septoria chrysanthemella and 5 for Septoria obesa. In
festation of the solid medium in petri dishes was accomplished by first
inserting a sterile needle into a speculating culture and then inserting
the needle vertically into the center of the medium. These cultures
were incubated for 2 days at 20C. and were then transferred to constant
temperature chambers at the temperatures given above, where they remained
for 28 days. The average diameter of each culture was then estimated by
making two measurements through the center of the culture. Four repli
cations of each fungus were used in the experiments.
The effects of different carbon sources on the growth of the
fungi on semi-synthetic agar medium was observed. Carbon sources used
in the experiments were mannose, D-mannitol, maltose, galactose, sucrose,
dextrose, and lactose; in addition there was a control group which lacked
a carbon source. All carbon sources were used at the rate of 10 g per
liter. The pH was adjusted to 5.0 after autoclaving. Four replications
were used for each fungus. The mean diameters were estimated as

39
previously described after Incubation of the cultures for 28 days at
20C.
The effects of several nitrogen sources on the growth of the
fungi in culture were studied. Semi-synthetic agar medium less biotin
and thiamine and with other nitrogen sources alternated with L-asparagine
was used. The quantity of each nitrogen source used was adjusted to
give a concentration of 425 mg of nitrogen per liter. The compounds
and quantities used per liter are given below:
L-asparagine 2.000 g
Glycine 2.277 g
L-leucine 3.977 g
L-arginine 1.321 g
Ammonium sulfate 2.014 g
Potassium nitrate 3.066 g
Controlnltrogen compounds omitted
Four replications for each fungus were used. The pH was ad
justed to 5.0 after autoclaving. Incubation was for 28 days at 20C.
Germination studies were made on pycnidiospores produced in
potato-dextrose-agar medium. The percentage of germination on water
agar at various temperatures was determined by observing 50 spores at
each temperature. Observations on pycnidiospore germination in several
kinds tt media were made.
RESULTS.The optimum pH for the growth of both Septoria obesa
and Septoria chrysanthemella in synthetic liquid medium was pH 5 to
pH 7. No growth occurred at pH 2 or pH 10, and little occurred at
pH 3 or pH 9. Summaries of the growth of S. obesa and S. chrysanthemel1a
are given in Tables 8 and 9y respectively.

40
tabu; s
THE EFFECT OF HYDROGEN-1 ON CQNCENTRATI ON ON THE GROTTO OF
SEPTORIA OBESA IN A SYNTHETIC LIQUID MEDIUM
pH
Dry Mycelial Weight
Range
Mean
Mean
Final pH
2
no growth occurred
3
45.0-57.9
53.4
3.3
4
41.6-81.7
54.6
4.5
5
98.0-169.5
129.6
6.9
6
109.8-133.7
123.9
7.1
7
73.4-125.2
99.0
7.2
8
46.0-71.1
60.2
7.7
9
31.4-40.0
36.2
8.6
10
no growth occurred
sl
After 28 days of growth,
b
Six replications were used.

41
TABLE 9
THE EFFECT OF HYDROGEN-ION CONCENTRATION ON THE GROWTH OF
SEPTOP.IA CIIRYSANTIIEMELLA IN A SYNTHETIC LIQUID MEDIUM
Dry Mycelial Weight in mga .
Mean
pH
Range
Mean
Final pH
2
no growth occurred
3
36.0-44.2
40.0
3.4
4
47.0-56.6
50.9
4.5
5
92.8-105.0
99.2
6.2
6
99.2-111.3
105.2
6.9
7
89.6-100.0
95.6
7.0
8
43.0-55.4
49.0
7.7
9
26.0-35.6
31.2
8.6
10
no growth occurred
aAfter 28 clays of growth,
b
Six replications wore used.

42
The amount of growth of Septoria obesa in synthetic liquid
media and on semi-synthetic agar medium at various temperatures is
shown in Tables 10 and 11, respectively. The results of similar experi
ments with Septoria chrysanthemella are shown in Tables 12 and 13.
S. obesa and S. chrysanthemella in culture on semi-synthetic agar
medium at optimum temperatures are shown in Fig. 12.
TABLE 10
THE EFFECT OF TEMPERATURE ON THE GROWTH OF SEPTORIA OBESA
IN SYNTHETIC LIQUID MEDIA
c
Dry Mycelial Weight
Range
in mga
Meanb
4
33.6-42.2
39.0
8
69.2-78.5
73.9
f O
102.0-114.2
109.5
16
12S.4-136.4
132.3
20
118.2-131.4
125.0
24
80.2-88.5
64.9
28
25.4-30.2
27.7
32
no growth occurred
aAfter 28 days of growth.
i-
Five replications were used.

43
TABUS 11
THE EFFECT OF TEMPERATURE ON THE GROWTH OF SEPTORIA OBESA
ON SEMI-SYNTHETIC AGAR MEDItBS
c
Culture Diameter
Range
in mma
Moan'0
Sporulation
4
2.5-3.5
3.0
no
8
5.56.5
6.0
yes
12
7.0-8.0
7.4
yes
16
10.5-12.0
11.0
yes
20
12.0-14.5
13.4
yes
24 V 'f
15.0-16.5
15.8
yes
28
12.2-14.4
13.2
yes
32 no growth occurred
aAfter 28 days of growth,
b
Four replications were used.

44
TABLE 12
TOE EFFECT OF TEMPERATURE ON THE GROWTH OF SEPTORIA
CHRYSANTIEMELLA IN SYNTHETIC LIQUID MEDIUM
Dry Mycelial Weight in mga
c
Range
Mean0
4
22.4-25.0
23.7
8
61.2-62.8
62.0
12
94.0-102.2
97.6
16' 1
105.7-121.4
113.1
20
93.4-109.0
100.6
24
71.8-78.6
75.6
28
61.0-64.5
62.7
32
no growth occurred
aAftor 28 days of growth.
|j
Three replications were used.

45
TABUS 13
THE EFFECT OF TEMPERATURE OH THE GROWTH OF SEPTORXA
CHRYSANTHEMELLA OH SEMI-SYNTHETIC AGAR 1KDIIB!
c
Culture Diameters
Range
in mma
Meaab
Sporulation
4
1.5-3.0
2.3
no
8
5.5-7.0
6.1
yes
12
10.5-12.0
11.3
yes
16
16.0-19.0
17.0
yes
20
23.5-28.5
26.0
yes
24
25.0-30.0
27.6
yes
28* '
31.5-35.5
33.5
yes
32 no growth occurred
aAfter 28 days of growth,
b
Four replications were used.

46
Fig. 12.Growth of Septoria obesa (left) and
Septoria chrysanthemella (right) on semi-synthetic
agar medium at optimum temperatures at the end of
40 days.

At temperatures of 4 to 20%. in the liquid medium, both of
the fungi produced black or greenish-black, compact masses of mycelium
which tended to adhere to the bottoms or sides of the vessels. At
24 and 23C. a colorless, film-like mycelium was formed which tended
to float on the surface of the liquid.
Temperature had no apparent effect on the color of cultures of
either fungus grown on the solid medium.
Both Septoria obesa and Septoria chrysanthemella sporulated on
all carbon sources used and also in the absence of carbon sources.
Data chi culture diameters are given in Tables 14 and 15.
TABLE 14
THE EFFECT OF CARRON SOURCE ON THE GROWTH OF SEPTORIA
OBESA ON SEMI-SYNTHETIC AGAR MEDIUM
Carbon
Culture Diameter
in mma
Source**
Range
Mean*3
Sporulation
Mannose
7.0-12.0
10.1
Yes
D-mannitol
7.0-8.5
8.0
Yes
Maltose
8.5-10.5
9.7
Yes
Galactose
8.5-11.5
10.5
Yes
Sucrose
9.0-11.5
9.9
Yes
Dextrose
10.0-11.0
10.4
Yes
Lactose
5.5-8.0
6.9
Yes
Minus carbon source
3.5-7.0
6.0
Yes
a
After 28 days of growth.
bAt the rate of 10 g per liter.
Four replications were used.

48
TABLE 15
THE EFFECT OF CARBON SOURCE ON THE GROWTH OF SEPTORIA
CHRYSANTHEf.SELLA ON SEMI-SYNTHETIC AGAR MEDIUM
Carbon
Source'3
Culture Diameter
Range
in mma
Mean0
Sporulatioa
Mannose
32.5-34.5
33.6
Yes
D-mannitol
25.0-27.0
26.0
Yes
Maltose
32.5-33.5
33.2
Yes
Galactose
35.0-37.0
36.1
Yes
Sucrose
35.5-43.5
40.6
Yes
Dextrose
35.5-37.0
36.2
Yes
Lactose
24.0-25.5
24.9
Yes
Minus carbon source
16.0-13.5
17.2
Yes
a
After 23 days of growth.
bAt the rate of 10 g per liter.
^our replications were used.
Septoria obesa cultures were very dark or black on all nitrogen
sources used and also on media lacking nitrogen; however, with glycine
and also with the absence of nitrogen, the cultures were less raised and
much less dense than with the other nitrogen sources. Pycnidiospores
were produced in every culture but were very sparse when glycine was
used and when nitrogen was lacking. The amount of growth of this fungus
on the various nitrogen sources is given in Table 16.

49
TABLE 16
THE EFFECT OF NITROGEN SOURCE ON THE GROWTH OF SEPTQRIA OBESA
ON SEMI-SYNTHETIC AGAR MEDIUM
Nitrogen
Source*1
Culture Diameter
Range
in imna
Mean0
Sporulation
Glycine
4.5-6.0
5.1
Yes
L-asparagine
11.5-13.5
12.8
Yes
L-leucine
4.5-6.0
5*0
Yes
L-arginine
8.09.5
8.9
Yes
Ammonium sulfate
7.0-3.0
7.5
Yes
Potassium nitrate
7.0-11.0
8.3
Yes
Minus nitrogen
5.56.5
6.1
Yes
aAfter 28 days of growth.
bAll nitrogen sources wore adjusted to give 425 ng of nitrogen
per liter.
CFour replications were used.
Septoria chrysanthcmsl1a grown on artificial media containing
different nitrogen sources varied markedly in both size and color. The
diameters of the cultures are given in Table 17. When L-asparagine was
used as a nitrogen source the culture was black, compact and raised, as
is typical of this fungus grown on potato-dextrose-agar. With glycine,
the cultures were conspicuously concentrically zoned with narrow dark
and light areas; zonation also occurred when L-arginine was used but was
not as distinct as with glycine. Potassium nitrate cultures were black

so
with small whitish areas dispersed near their centers. With L-leucine
a white culture with dispersed black spots resulted and ammonium sulfate
gave a white fluffy culture with a greenish center. Cultures which
lacked nitrogen had yellow interiors which were covered by a white
mycelium. No pycnidiospores were produced when ammonium sulfate was
used or when nitrogen was lacking, and very few were formed when glycine
was used. Cultures of the fungus growing on different nitrogen sources
are shown in Fig. 13.
TABUS 17
the effect of nitrogen source on the growth of septoria
CHRYSANTHEMELLA ON SEMI-SYNTHETIC AGAR MEDIUM
Nitrogen
Source*5
Culture Diameter
Range
in mma
Meanc
Sporulation
Glycine
14.5-18.5
15.8
Very little
L-asparagine
32.5-33.5
33.3
Yes
L-leucine
20.0-22.0
21.0
Yes
L-arginine
28.0-27.0
26.6
Yes
Ammonium sulfate
17.0-21.5
18.9
No
Potassium nitrate
19.5-24.0
22.0
Yes
Minus nitrogen
15.0-16.5
15.6
No
a
After 28 days of growth,
b
All nitrogen sources were adjusted to give 425 mg of nitrogen
per liter,
c
Four replications were used.

51
Fig, 13.Growth characteristics of Septoria
chrysantheraella on serai-synthetic agar medium sup
plied with different nitrogen sources (1) L-asparagine,
(2) L-arginine, (3) glycine, (3) potassium nitrate,
(5) ammonium sulfate, (6) L-leucine, (7) minus nitrogen.

52
Both Septoria obesa and Septoria chrysaathemella grow slowly on
potato-dextrose-agar medium. Both cultures are black, raised, rough-
surfaced and compact. S. obesa usually makes more vertical growth, and
S. chrysanthemella spreads more rapidly on the surface of the medium.
The manner of germination of pycnidiospores of Septoria obesa
and Septoria chrysanthemella on water agar differs fresa that of spores
submerged in water. On water agar and on potato-dextrose-agar medium
the spores swell, leaving constrictions at the septa and frequently
between the septa. The first germination hyphae formed usually extend
axially from the basal and apical cells of the spores but lateral hyphae
arise a few hours later from other cells of the spores (Fig. 14).
Pycnidiospores germinating in water, potato-dextrose broth, or synthetic
liquid media do not swell greatly or become conspicuously constricted,
and they germinate mainly by hyphae extending axially from the basal
and apical cells (Fig. 15). Occasionally a pycnidiospore germinating
in water will produce lateral hyphae during the early stages of germina
tion, but this usually does not occur until germination is well advanced
and the spore has become almost indistinguishable from the mycelium.
There is also a difference in the rate of germination of pycnidio
spores in liquids and on solid media. Septoria chrysanthemel1a pycnidio
spores on water agar at 20C. begin to germinate in 7 hours, and there
may be 100 per cent germination at the end of 24 hours. In water,
germinated pycnidiospores of this fungus are rare at the end of 24 hours
and the percentage of germination is usually 50 or less at the end of

53
Fig. 14.Pycnidiospores of Septoria ehrysanthe-
mella germinating on solid medium Fig. 15.Pycnidiospores of Septoria chrysanthe
me! la germinating in water (X 1000).

43 hours* Pycnidiospores of Soptoria obesa on water agar at 20C*
54
may germinate in very low percentage after 5 hours, and the percentage
of germination may bo almost 100 at the end of 24 hours* One hundred
per cent germination of spores of this fungus was not observed, even
after 5 days on water agar. The germination rate in water is as de
scribed for Septoria chrysanthemella.
The percentage of germination of pycnidiospores of the fungi on
water agar at various temperatures is given in Table 13.
tabie IS
HERCENTA OF GERMINATION OF PYCNIDIOSPORES ON WATER
AGAR AT VARIOUS TEMPERATURES
c
S. dhry s anthemel1a
Time in Hours
S* obesa
Time in Hours
10
20
48
10
20
48
4
0
0
0
0
0
0
3
0
0
76
0
10
84
12
0
6
88
0
16
92
16
8
92
98
4
90
96
20
18
96
96
20
96
98
24
16
100
0
20
96
94
23
52
100
0
16
94
96
32
0
0
0
0
0
0

DISCUSSION.The optimum pH for the growth of both Septoria
obesa and Septoria chrysanthemella was found to be from pH 5 to pH 7, \D
These findings are not exceptional, as most fungi which have been
investigated grow best in slightly acid media.
Both fungi made relatively poor growth in liquid medium at 24 ^
and 28C. whereas maximum growth on solid medium was at these same
temperatures. The poor growth at the higher temperatures in liquid
medium could possibly be related to the lower retention of oxygen and
other gases by liquids at higher temperatures, or chemical changes un
favorable to the fungi may have occurred at higher temperatures. Neither
fungus grew at 32C. With regard to Septoria chrysanthemella, this is
in conflict with the findings of Heneni and Nakamura (22) that this
fungus made better growth at 32C. than at 20C. This difference could
be due to experimental errors or to slight physiological differences
between the American isolates and the Japanese isolates.
Each of the two fungi grew and sporulated on all carbon sources
used and also in the absence of a carbon source. Growth of both of the
species was poorest when carbon was lacking and when lactose was used
as a carbon source. There was apparently very little difference in the
rate of growth of either of the fungi on the several other carbon sources
used.
Both Septoria obesa and Septoria chrysanthemella grew more
rapidly when L-asparagine was used as a nitrogen source than when other
nitrogen sources were used. The growth of both fungi was relatively slow
on glycine, ammonium sulfate, and in the absence of nitrogen. Septoria

36
chrysanthemel1a was sensitive to the type of nitrogen source, exhibit
ing various colors and patterns in culture and failing to produce
pycnidiospores in the absence of nitrogen or with ammonium sulfate.
Whether or not the nitrogen source requirements of this fungus are
related to its selective pathogenicity on chrysanthemum varieties has
not been investigated,
Hiere was little or no difference in the rate or manner of
germination of the pycnidiospores of the two fungi in water or on
solid media.

37
Pathogenicity
In numerous inoculation experimente all isolates of Septoria
obesa and all isolates of Septoria chrysanthemella produced character
istic disease symptoms on certain chrysanthemum varieties, whereas
check plants remained healthy. In all cases, pycnidiospores character
istic of the Septoria species involved were abundant in the disease
lesions of the host foliage.
It has been reported by Gram and Weber (17), Raff (30), Bohmig
(3), and Fischer (16) that chrysanthemum varieties vary in suscepti
bility to septoria leafspot. However, all of these reports were based
on observations and none were made under controlled conditions by
artificial inoculations.
The pathogenicity of Septoria obesa and Septoria chrysanthe-
mslla on twenty-two varieties of chrysanthemum were investigated in
a series of inoculation experiments conducted under variable conditions
in the greenhouse and in the field.
METHODS AND MATERIALS.All plants in the greenhouse were grown
in 4-inch, clay pots containing soil which had previously been dis
infected with methyl bromide. Hie soil in outdoor plots was not treated;
however, no chrysanthemum had been grown in the immediate vicinity for
several years. Fungicides were not used on experimental plants, but
propagation stock was treated with a ferbam spray about once a week.
Malathioa or nicotine sulfate sprays were used as needed for insect
control on all plants

58
Plants in the greenhouse experiments were inoculated by
atomizing both surfaces of all leaves with spore suspensions made
from pure cultures of the isolates. Immediately after inoculation the
plants were placed in a mist chamber for S days and then removed to a
greenhouse bench. The positions of the plants in the mist chamber and
on the greenhouse beach were always randomized. A control group con
sisting of one plant of each variety used in the particular experiment
was not inoculated with spores of these fungi but otherwise received
the same treatment as did the inoculated plants.
Plants in experiments conducted in outdoor plots were inoculated
with spore suspensions applied with a pressure sprayer. Control plants
were covered by plastic bags during the inoculation operation.
In evaluating the severity of the disease produced by Septoria
chrysanthemella oily the number of lesions formed was considered. Spots
produced by this fungus seldom exceeded one centimeter in diameter. Hie
scale used considered the average number of spots per leaf on the lower
four leaves. Four classes were used in this rating as follows:
Severe: Average of 8 or more spots per leaf.
Moderate: Average of 3 to 7 spots per leaf.
Mild: Average of 1/4 to 2 spots per leaf.
Healthy: Ho infection.
Hie severity of the disease caused by Septoria obesa cannot be
accurately judged by the number of lesions per leaf, as single spots
frequently cover one-quarter or more of a leaf. After observing the
disease produced in the first inoculation experiment the following
scale was formulated which subsequently proved to be applicable in all
other inoculation experiments with S. obesa:

59
Severe: Most of the inoculated leaves infected.
Mild: Only the lower leaves infected.
Healthy: No infection.
Other details of methods employed are given below for each
experiment. The experiments are not necessarily numbered in chrono
logical order.
Experiment 1.Four plants each of 15 chrysanthemum varieties
propagated in the greenhouse the previous spring were removed from pots
in August, 1958, and randomized in a garden plot. The plot consisted
of 4 rows 3 feet apart, and plants in rows were spaced 18 inches apart.
Three plants of each variety were inoculated on September 10 with
isolate No. 1 of Septoria obesa. The plants were kept wet by a
sprinkler for 3 days following inoculation. Disease severity was
evaluated 5 weeks later.
Experiment 2.Four plants each of 22 chrysanthemum varieties
in the flowering stage were inoculated in two groups of 11 varieties
each in the greenhouse on October 15 and 18, 1958, with isolate No. 1
of Septoria obesa. Disease severity was evaluated 6 weeks later.
Experiment 3.Four young plants each of 22 chrysanthemum
varieties were inoculated in the greenhouse in two groups of 11 varieties
each with isolate No. 2 of Septoria obesa on March 13 and 16, 1959. All
plants were rooted cuttings with 4 to 6 expanded leaves on each plant.
Disease severity was evaluated 5 weeks later.
Experiment 4.Four plants each of 22 chrysanthemum varieties
in the flowering stage were inoculated in two groups of 11 varieties
each in the greenhouse on November 9 and 12, 1958, with isolate No. 1
of Septoria chrysanthemella. Disease severity was evaluated 6 weeks
later

60
Experiment 5.Four young plants each of 22 chrysanthemum
varieties were inoculated in two groups of 11 varieties each with
isolate No. 2 of Septoria chrysanthemel1a on March 6 and 9, 1959.
All plants were rooted cuttings with 4 to 6 expanded leaves on each
plant. Disease severity was evaluated 5 weeks later.
Experiment 6.Five plants each of 14 chrysanthemum varieties
were removed from pots and planted with 1-foot spacings in a garden
plot early in April, 1959. On April 15, four plants of each variety
were inoculated with isolate No. 4 of Septoria chrysanthemella, and
the plants were kept wet for 3 days afterwards by means of a sprinkler.
The plants were six weeks old when inoculated. The control group
consisted of 1 plant each of the varieties. Disease severity was
evaluated 5 weeks after inoculation.
RESULTS.Experiment 1.All chrysanthemum varieties artificially
inoculated in the field with Septoria obesa became severely diseased.
Most of the control plants had a few lesions, but these were confined
to leaves near the soil. The data are given in Table 19.
Experiments 2 and 3.Young and old plants in the greenhouse were
severely infected by Septoria obesa, irrespective of the chrysanthemum
variety. Data on these two experiments are summarized in Table 20.
Experiments 4 and 5.Septoria chrysanthemel1a attacked young
and old plants in the greenhouse with equal severity, but there were
definite differences in the pathogenicity of this fungus on various
chrysanthemum varieties. Data are shown in Table 21.
Experiment 6.There were marked differences in disease severity
on different chrysanthemum varieties artificially inoculated in the
field with Septoria chrysanthemella. Data are shown in Table 22.

61
TABLE 19
SEVERITY OF DISEASE ON MATURE PLANTS OF FIFTEEN CHRYSANTHEMUM
VARIETIES INOCULATED WITH SEPTORIA OBESA IN THE FIELD
Chrysanthemum No. of No. of Plants in Each Disease Class
Variety Plants Severe Mild Healthy
Alaska 3 3
Blue Chip 3 3
Copperhead 3 3
Dark Buckley 3 3
Illini Igloo 3 3
Illini Regal 3 3
Indianapolis Bronze 3 3
Jetfire 3 3
Mary L. Hall 3 3
Pink Dot 3 3
Portrait 3 3
Rayonnante 3 3
Seneca 3 3
White Top 3 3
Yellow Shasta 3 3
Control Plants
15
10
5

62
TABLE 20
SEVERITY OF DISEASE ON TWENTY-TWO CHRYSANTHEMUM VARIETIES
INOCULATED WITH SEPTQRIA OBESA IN THE GREENHOUSE
Chrysanthemum No. of No. of Plants in Each Disease Class
Variety Plants Severe Mild Healthy
Alaska 8
Beauregard 8
Blue Chip 8
Bonnie 8
Copperhead 8
Dark Buckley 8
Delaware 8
Gold Ball S
Humdinger 8
Illini Igloo 8
Illini Regal 8
Illini Warpaint 8
Indianapolis Bronze 8
Jetfire 8
Mary L. Hall 8
Pink Dot 8
Portrait 8
Rayonnante 8
Seneca 8
White Top 8
Yellow Queen 8
Yellow Shasta 8
Control Plants 44
8
8
4
7
8
8
8
7
7
8
6
7
7
7
7
6
8
7
8
8
8
8
4
1
1
1
1
1
1
1
2
1
1
1
3
41

63
TABLE 21
SEVERITY OF DISEASE ON TWENTY-TWO CHRYSANTHEMUM VARIETIES INOCULATED
WITH SEPTORIA CHRYSANTI IE MELLA IN THE GREENHOUSE
Chrysanthemum No. of No. of Plants in Each Disease Class
Variety Plants Severe Moderate 1 Mild Healthy
Alaska 8
Beauregard 8
Blue Chip 8
Bonnie 8
Copperhead 8
Dark Buckley 8
Delaware 8
Gold Ball 8
Humdinger 8
Illini Igloo 8
Illini Regal 8
Illini Warpaint 8
Indianapolis Bronze 8
Jetfire 8
Mary L. Hall 8
Pink Dot 8
Portrait 8
Rayonnaate 8
Seneca 8
White Top 8
Yellow Queen 8
Yellow Shasta 8
Control Plants 44
1
7
8
1
5
4
2
1
6
3
1
1
6
1
7
2
3
4
5
3
2
4
5
4
4
2
4
3
6
3
2
2
6
4
1
2
3
5
1
3
4
4
6
3
5
2
4
5
2
4
44

64
TABLE 22
SEVERITY OF DISEASE ON FOURTEEN CHRYSANTHEMUM VARIETIES INOCULATED
WITH SEPTORIA CHRYSANTHEMELLA IN THE FIELD
Chrysanthemum
Variety
No. of No. of Plants in Each Disease Class
Plants Severe Moderate Mild Healthy
Alaska 4
Beauregard 4
Bonnie 4
Dark Buckley 4
Delaware 4
Gold Ball 4
Illini Regal 4
Indianapolis Bronze 4
Jetfire 4
Mary L. Hall 4
Portrait 4
Rayonnante 4
White Top 4
Yellow Shasta 4
Control Plants 14
2
3
1
2
2
2
3
3
1
2
1
1
1
2
2
1
1
2
2
2
1
2
1
1
5
1
3
1
2
1
4
2
1
9

es
DISCUSSION.Septoria obesa was very pathogenic on both young
and old plants of all of the 22 chrysanthemum varieties inoculated.
Whether or not any of the several thousand chrysanthemum varieties grown
today possess any degree of resistance to S. obesa cannot be predicted.
However, it is reasonable to suppose that a large majority of chrysan
themum varieties is susceptible to this pathogen.
Septoria chrysanthemella infected young and old plants of the
same chrysanthemum variety equally, but the pathogenicity of this fungus
varied greatly on different chrysanthemum varieties. The varieties
Mary L. Hall and Bonnie alone were consistently, severely infected,
whereas a few others were never more than mildly infected. There is
apparently no doubt that the pathogenicity of S. chrysanthemella varies
on different varieties of the cultivated chrysanthemum.
Septoria obesa is more pathogenic than Septoria chrysanthemella.
The former species severely attacked all chrysanthemum varieties in
oculated, and even a few lesions an a leaf frequently enlarged greatly,
killing the entire leaf. Not only was S. chrysanthemella only mildly
pathogenic on many chrysanthemum varieties, but lesions on susceptible
plants were usually less than 1 cm in diameter, and leaves were not
killed unless the lesions were very numerous.

HOST-PARASITE RELATIONS
Seasonal Development of the Diseases
The development of the disease caused by Septoria obesa has
been observed for the past 13 months in unprotected chrysanthemum
plantings in Gainesville, Florida. In a field plot on which chrysan
themums were heavily diseased the previous fall, young shoots developed
symptoms about two weeks after emerging from the soil in February and
March.
Once infection has occurred, the plants remain diseased until
killed by freezes the following winter. The development of symptoms
and the spread of the disease are affected more by climatic conditions
than by the season; however, in general, the development of symptoms
and the spread of the disease are more rapid in the spring and fall
than in the summer.
The seasonal development of the disease caused by Septoria
chrysanthemella was not observed, but various reports in literature
indicate that its seasonal development is very similar to that of
Septoria obesa.
Sources of the Inoculum
The source of primary inoculum in the field is the pycnidio-
spores in pycnidia imbedded in diseased leaves remaining on the ground
in the fall and winter when plants are not growing. Dimock and Allen
(13) observed the disease develop late in the summer in cut-over beds
where septoria leafspot had been controlled during the earlier part of
66

c?
tli year by spraying; they suggested that the inoculum was carried
over from the previous fall.
An experiment was undertaken to determine the longevity of
the inoculum on and in the soil in the Gainesville area. Field con
ditions wore simulated as nearly as possible. The dry, lower leaves
of chrysanthemum plants heavily infected with Septorla obesa were
talcen late in November and crumbled and mixed into field soil. This
mixture was then used to form the upper inch of soil in three 5-inch
pots previously two-thirds filled with field soil from another location.
The pots were then moved to a location remote from chrysanthemum plant
ing and placed in the ground with surfaces of the potted soils at
ground level. The same procedure was followed with inoculum from
Septorla chrysanthemella except that diseased leaves from the green
house were used and the operation was not begun until the middle of
December, On February 4, leaf fragments were examined, and spores of
both of the fungi were found by microscopic examination. On March 3,
spores were still present and germinated well on water agar.
An experiment was then designed to test the pathogenicity of
the spores of each of the fungi. Three young plants of variety Jetfire
were inoculated by dipping into a water suspension of the Septorla
obesa spores. Two sets of three each of control plants were used, all
of variety Mary L. Hall, One control group was dipped into a suspension
of field soil collected at a location remote from chrysanthemum plant
ings, The other control group was inoculated with a water suspension
of S. obesa spores from culture. After inoculation all plants were
maintained in a mist chamber for 3 days and then were removed to a

68
greenhouse bench. The same procedure was followed, and at the same time,
with Septoria chrysanthemella except that the chrysanthemum variety used
was Mary L. Hall.
All of the plants inoculated with the suspension containing the
diseased leaf fragments became infected. Disease lesions were not
numerous on these plants, but pycnidiospores characteristic of the species
of Septoria involved were eventually produced. None of the plants dipped
into the suspension of field soil became infected. All plants inoculated
with spore suspensions from culture became heavily infected.
On April 5 no spores could be found in the over-wintered inoculum.
However, the inoculation experiment as described above was repeated, but
the only plants that became infected were those inoculated with spores
from culture.
It appears to be very doubtful that inoculum in diseased leaves
left on the soil in the fall remains viable into the late spring or into
the summer in the Gainesville area.
After plants have become infected, pycnidia and pycnidiospores
are formed in the leaf lesions. The pycnidiospores produce secondary
infection when disseminated to other leaves or other plants if conditions
are favorable.
Spore Dissemination
It has been noted by many observers that new infections of
chrysanthemum plants by septoria diseases follow periods of rainy
weather. According to Dimock (10) the pycnidiospores are disseminated
by splashed or wind-blown water, or by various mechanical means when

the foliage is wet. He found that direct splashing of spores from the
soil may reach a height of 16 to 18 inches. Dimock (11) also found
that the spores were disseminated by insecticidal sprays and that most
insecticides commonly used have little or no effect on the viability
of the spores.
Climatic Conditions Affecting Infection and Disease Development
According to Dimock (10), pycnidiospores must be in contact
with the wet abaxial surface of a leaf for at least 24 hours before
infection is established; the 24-hour wet period does not have to be
continuous for infection to occur but may alternate with dry periods
such as may occur with nightly dews and sunny days.
Hie writer agrees that some infection by each of the Septoria
species may occur after a 24-hour wet period for the pycnidiospores on
the abaxial surface of the leaf. However, several experiments in which
inoculated plants were maintained in a mist chamber for only one day
resulted in little or no infection. Furthermore, only an occasional
germinated spore of either fungus was observed on cleared leaves which
had previously been inoculated and kept wet for 24 hours at 20C; even
after 43 hours only about one-half of the spores had germinated.
Extremely hot weather retards the development of the diseases,
both in the field and the greenhouse. All inoculation experiments
carried out in the greenhouse during the hot summer months either failed
completely, or else the development of symptoms was arrested until a
period of mild weather arrived. In one case plants inoculated with
Septoria obesa did not show disease symptoms until 6 weeks later. The

70
day temperatures in the greenhouse during sunny summer days usually
ranged between 85 and 95F. During mild weather symptoms of both
diseases usually become visible in 12 to 18 days and pycnidiospores
are present about 1 week later* This agrees with Dimock (10) who
gives the incubation period at 10 to 14 days, with spores developing
4 to 6 days later.
It was noted by Halsted (20) in 1891 that moisture greatly
favors the development of septoria leafspots, and this has been
frequently observed since that time. Not only are the spores dis
seminated during rainy periods, causing new infections, but lesions
already present increase in size.
Host Penetration
According to Dimock (10, 12) infection by Septoria obesa or
Septoria chrysaathemella occurs almost exclusively at the abaxial sur
faces of the leaves. In several inoculation experiments carried out
with plants under bell jars over water* the writer found this to be
true; only rarely did infection occur when only the adaxial surfaces
of leaves were inoculated. However, literature reveals no information
on the specific mode of host penetration by either fungus; consequently,
a study of this was undertaken*
METHODS AND MATERIALS.After much experimentation with various
leaf-clearing and staining techniques, the best method, and also the
most rapid, was found to be a combination of the method used by Peace
(29) to clear dead leaves and a technique described by Diener (9) for
staining fungus spores and hyphae on leaves.

71
Equal volumes o crystals of chloral hydrate and phenol were
placed in a beaker and heated gently until melted; a snail vial was
then one-half filled with a portion of this mixture.
One-centimeter squares were then cut from leaves of susceptible
chrysanthemum plants previously heavily inoculated and maintained under
bell jars over water for 3 to 5 days. Several squares of leaf tissue
were then placed into the mixture in the vial and heated to near the
simmering point until the tissue became translucent. This usually
required les3 than 30 minutes. The leaf tissue was then soaked in warm
lacto-phenol for at least 15 minutes. A square of the tissue was then
placed on a glass slide, flooded with 1acto-phenol deeply colored by
a 1 per cent cotton blue solution, and heated to the simmering point
for 2 or 3 minutes. The material was then examined under the micro
scope, and if staining was inadequate the slide was heated again. The
slide was then flooded with unstained lacto-phenol, and a cover slip
was placed over the material. When not being studied, the leaf material
was stored in lacto-phenol.
OBSERVATIONS AND DISCUSSION.Numerous observations were made
in tills study, both on the abaxial and adaxial surfaces of the inoculated
leaves. The spores germinated in the manner and apparently at about
the same rate as previously described in the section on physiology for
the germination of spores in water.
On the abaxial surface, germination hyphao frequently enter
stomata; however, the hyphae apparently are not attracted to stomata,
as they much more frequently passed directly over them. No evidence
of penetration directly through epidermal cells was observed.

Although the ataxial epidermis of chrysanthemum leaves has a
few scattered stomata, penetration vas not observed. However, it Is
not unreasonable to suppose that penetration through the stomata of
this epidermis occasionally occurs.
Penetration of stomata by Septoria obesa and Sep tori a chrysaa-
theaella hyphae are shown in figs. 16 and 17, respectively.
Pathological Anatomy
Morphological characters of the myeelia and pycnidia of the
two pathogens in the leaves of the host have already been described
in this paper. The study given below is concerned only with the
anatomical relationships of fungous and host tissues,
METHODS AND MATERIALS. -Observations are based an the study of
permanent slides of lesions and adjacent tissues of host leaves. The
leaf material ms killed mad fixed for at least 48 hours in formalin-
acetic-alcohol <10 parts 95 per cent ethanol, 1 part glacial acetic
acid, 2 parts formalin, 7 parts water) and embedded in paraffin.
Serial sections 10 microns thick were made with a rotary microtome.
The sections were then fixed to glass slides with Kaupt's adhesive and
either left unstained or stained with safranin, safranin and fast green,
iron-hematoxylin, or cotton blue. In general safranin was best for
staining the mycelium, whereas pycnidia were best observed unstained.
All sections wore mounted in balsam.
OBSERVATIONS AND DISCUSSION.The invasion of healthy host
tissues by the mycelia of Septoria obesa and Septoria chryaanthemella
is apparently identical. The mycelium grows intereellularly, surrounding

Fig. 16.Stomatal penetration by a hypha from
a germinating pycnidiospore of Septoria obesa (X 476)
Fig. 17.Stomatal penetration by a hypha from
a germinating pycnidiospore of Septoria chrysanthemella
(X 476).

74
the host cells and causing their death. No evidoace of direct
penetration or haustoria formation was observed. The mycelium grows
most freely in the spongy and palisade mesophyll but also invades the
bundle sheaths and vascular tissues. The mycelium was observed to
extend as much as 2 mm from the necrotic tissue of the lesion.
Figure 18 shows the mycelium of Septoria obesa surrounding cells of
the mesophyll. *
Hie mycelium surrounding lysigenous cavities adjacent to
epidermal tissues sometimes forms numerous small, spherical or
elliptical-shaped cells. Hiis is apparently an early stage in the
formation of a pycnidium. Pycnidia were present only in necrotic
tissue in this material but have been observed in adjacent chlorotic
tissues in untreated leaves. Short pycnidial beaks rupture the epidermis
and extend above it or sometimes extend through stomata.
Host tissue invaded by Septoria obesa and Septoria chrysanthemella
are shown in Figs. 19 and 20, respectively.

75
Fig. 18.-Cross-section of host leaf showing
intercellular invasion of mesophyll tissue by Septoria
obesa mycelium (X 476).

Fig. 20.--Cross-section through a leaf lesion
showing pycnidia of Septoria chrysanthemel1a (X 111)

SUMMARY
1* Two distinct septoria diseases of the cultivated chrysanthemum
are common in the United States and in several other parts of
the world where the host is grown.
i
2. The valid names of the causal organisms are Septoria obesa Syd.
and Septoria chrysanthemella Sacc.
3. The disease caused by Septoria obesa appears to be more common
in the United States and is also more severe than the disease
caused by Septoria chrysanthemella.
4. Symptoms of each disease are described in detail. Septoria obesa
caused black or dark brown leaf lesions which are circular to
irregular in shape and indefinite in size. Septoria chrysanthe
mella causes black, circular leaf lesions which are usually one
centimeter or less in diameter.
5. Hie morphology of the causal organisms in culture and in host
tissues is described in detail. There is a distinct difference
in average size, shape, and number of septa of the pycnidiospores
of the two pathogens. Geographical location or host variety did
not significantly affect pycnidiospore morphology.
6. The optimum hydrogen-ion concentration for the growth of each
fungus in culture was pH 5 to pH 7; no growth occurred at pH 2
or pH 10.
7. The optimum temperatures for the growth of each pathogen in
culture on solid medium was 20 to 28C. Little growth occurred
at 4C., and there was no growth at 32C.
8. The rate and character of growth of the pathogens in culture on
various carbon sources and nitrogen sources is described.
9. Pycnidiospores germinated rapidly on solid media and germinated
slowly in water. The rate of germination at various temperatures
is described.
10.Septoria obesa severely attacked all chrysanthemum varieties
inoculated, but the pathogenicity of Septoria chrysanthemella
varied greatly on different varieties of the host.
77

7S
11. In the Gainesville, Florida, area symptoms of the disease may
be present on chrysanthemum leaves which are two or more weeks
old during any season of the year.
12. Inoculum in diseased leaves left on and in the soil in December
did not remain viable into April of the following spring.
13. Pycnidiospores of the causal organisms are disseminated princi
pally by spattering water. Infection progresses from the lower
leaves upward to the upper leaves of the plants.
14. Infection and disease development are favored by moisture and
cool or mild temperatures and are restricted or retarded by very
hot, dry weather.
15. Penetration of the host is by hyphae of germinated spores enter
ing stomata of the leaves.
16. Pathological anatomy is described in detail. The mycelium of
each of the causal organisms grows intercellularly and does not
produce haustoria.

LITERATURE CITED
1. Beach, B. S, 1893. Leaf-spot of chrysanthemums. Eleventh
Ann. Rept. N. Y. Agr. Exp. Sta. (Geneva), p. 557.
2. Bensande, M. 1927. Inventario das molestias das plantas
agricolas de S. Miguel. (Abs.) Rev. Appl. Mycol. 6:466.
3. Bohmig, F. 1937. Uber die Anfalligkeit der Chrysanthemum-
Sorten Gartenwelt 41:510.
4. Brien, R. M. and Joan M. Dingley. 1953. First supplement to
"a revised list of plant diseases recorded in New Zealand."
(Abs.) Rev. Appl. Mycol. 33:53.
5. Chifflot, J. 1904. Maladies et Parasites du Chrysantheme.
Librairie et Imprimerie Horticoles. Paris. 56 pp.
6. Cooke, M. C. 1887. New British fungi. Grevillea 15:109.
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8. De Carvalho, T. 1948. Relacao preliminar de doencas encontradas
em plantes e insectos com anatacoes fitopatologicas. (Abs.)
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9. Diener, U. L. 1955. Host-penetration and pathological histology
in gray leaf spot of tomato. Phytopathology 45:654-658.
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11. Dimock, A. W. 1951. The dispersal of viable fungus spores by
insecticides. Phytopathology 41:152-156.
12. Dimock, A. W. 1953. Control of three ills of chrysanthemum.
Plant Diseases Yearbook of Agriculture. U. S. Dept. Agr.
Washington, pp. 592-595.
13. Dimock, A. W. and H. Allyn. 1944. Dipping rooted chrysanthemum
cuttings in fermate for septoria leafspot control. Bull. Chrysanth.
Soc. Amer. 12:9-11.
14. Eastham, J. W. 1937. Report of Provincial Plant Pathologist.
(Abs.) Rev. Appl. Mycol. 16:797.
79

00
15. Ellis, J. B. and J. Dearness. 1893. New species of Canadian
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27. Mata Quesada, E. 1950. Principles enfermedades y plagas en los
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28. Niklova, H. 1949. Tri ctvrti roku zahradnicke praxe ocima
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81
31.
Saccardo,
P.
A.
1884.
Sylloge
fungorum 3:549.
32.
Saccardo,
P.
A.
1895.
Sylloge
fungorum 11:542.
33.
Saccardo,
P.
A.
1898.
Sylloge
fungorum 14:973.
34.
Saccardo,
P.
A.
1913.
Sylloge
fungorum 22:1104.
35.
Salmon, E. S
(Abs.) Rev.
, and W. M. Ware. 1937. Department of mycology.
Appl. Mycol. 16:366.
36.
Sydow, H.
and P.
Sydov.
1914.
Zweiter Beitrag zur Kenntnis der
parasitischen Pilzflora des nordlich Japans. Asnales Mycologici
12:184.
37. Trent, J. A. 1939. The status of Cylindrosporium chrysanthemi
S. & D. as the causative agent of chrysanthemum leaf blight.
Trans. Kans. Acad. Sci. 42:203-204.
38. Voglino, P. 1901. Sopra una malattia dei crisantemi coltivati.
Malpighia 15:329.
39. Weiss, F. 1950. Index to plant diseases in the United States.
U. S. Dep. Agr. Plant Disease Survey Special Pub. 1, part 1.
p. 169.

BIOGRAPHICAL SKETCH
Henry Thomas Waddell was born in Wilson, Arkansas, on
April 19, 1918. He attended public schools in Arkansas and
Tennessee and graduated from Troy High School, Troy, Tennessee,
in 1937. He served for six years with the 30th Infantry Bivision
during World War II and was separated from service in 1946 with
the rank of 1st Lieutenant.
Mr. Waddell completed two years of study at the University
of Tennessee Junior College, Martin, Tennessee, and continued his
studies at George Peabody College for Teachers, Nashville, Tennessee,
where he received the Bachelor of Science degree in 1949 and the
Master of Arts degree in 1951.
He was employed as Assistant Professor of Biology by the
University of Tennessee Martin Branch, Martin, Tennessee, from
September, 1949, to June, 1956.
Mr. Waddell entered the University of Florida in June, 1956,
to pursue a course of study leading to the Doctor of Philosophy degree
with a major in Plant Pathology.
He is a member of the American Phytopathological Society, the
American Institute of Biological Sciences, Gamma Sigma Delta, and
Plii Kappa Phi.
82

This dissertation was prepared under the direction of
the chairman of the candidate's supervisory committee and has
been approved by all members of that committee. It was sub
mitted to the Dean of the College of Agriculture and to the
Graduate Council, and was approved as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.
August S, 1959
OaaA. Dean, College of Agriculture
Dean, Graduate School
SUPERVISORY COMMITTEE:



62
TABLE 20
SEVERITY OF DISEASE ON TWENTY-TWO CHRYSANTHEMUM VARIETIES
INOCULATED WITH SEPTQRIA OBESA IN THE GREENHOUSE
Chrysanthemum No. of No. of Plants in Each Disease Class
Variety Plants Severe Mild Healthy
Alaska 8
Beauregard 8
Blue Chip 8
Bonnie 8
Copperhead 8
Dark Buckley 8
Delaware 8
Gold Ball S
Humdinger 8
Illini Igloo 8
Illini Regal 8
Illini Warpaint 8
Indianapolis Bronze 8
Jetfire 8
Mary L. Hall 8
Pink Dot 8
Portrait 8
Rayonnante 8
Seneca 8
White Top 8
Yellow Queen 8
Yellow Shasta 8
Control Plants 44
8
8
4
7
8
8
8
7
7
8
6
7
7
7
7
6
8
7
8
8
8
8
4
1
1
1
1
1
1
1
2
1
1
1
3
41


61
TABLE 19
SEVERITY OF DISEASE ON MATURE PLANTS OF FIFTEEN CHRYSANTHEMUM
VARIETIES INOCULATED WITH SEPTORIA OBESA IN THE FIELD
Chrysanthemum No. of No. of Plants in Each Disease Class
Variety Plants Severe Mild Healthy
Alaska 3 3
Blue Chip 3 3
Copperhead 3 3
Dark Buckley 3 3
Illini Igloo 3 3
Illini Regal 3 3
Indianapolis Bronze 3 3
Jetfire 3 3
Mary L. Hall 3 3
Pink Dot 3 3
Portrait 3 3
Rayonnante 3 3
Seneca 3 3
White Top 3 3
Yellow Shasta 3 3
Control Plants
15
10
5


Although the ataxial epidermis of chrysanthemum leaves has a
few scattered stomata, penetration vas not observed. However, it Is
not unreasonable to suppose that penetration through the stomata of
this epidermis occasionally occurs.
Penetration of stomata by Septoria obesa and Sep tori a chrysaa-
theaella hyphae are shown in figs. 16 and 17, respectively.
Pathological Anatomy
Morphological characters of the myeelia and pycnidia of the
two pathogens in the leaves of the host have already been described
in this paper. The study given below is concerned only with the
anatomical relationships of fungous and host tissues,
METHODS AND MATERIALS. -Observations are based an the study of
permanent slides of lesions and adjacent tissues of host leaves. The
leaf material ms killed mad fixed for at least 48 hours in formalin-
acetic-alcohol <10 parts 95 per cent ethanol, 1 part glacial acetic
acid, 2 parts formalin, 7 parts water) and embedded in paraffin.
Serial sections 10 microns thick were made with a rotary microtome.
The sections were then fixed to glass slides with Kaupt's adhesive and
either left unstained or stained with safranin, safranin and fast green,
iron-hematoxylin, or cotton blue. In general safranin was best for
staining the mycelium, whereas pycnidia were best observed unstained.
All sections wore mounted in balsam.
OBSERVATIONS AND DISCUSSION.The invasion of healthy host
tissues by the mycelia of Septoria obesa and Septoria chryaanthemella
is apparently identical. The mycelium grows intereellularly, surrounding


LIST OF TABLES
Table Page
1. Morphological Characters of Septoria obesa as
Reported by Different Investigators . 16
2. Morphological Characters of Septoria chrysanthemella
as Reported by Different Investigators ... 17
3. Dimensions and Number of Septa of Pycnidiospores and
Diameters of Pycnidia of Septoria obesa Specimens
Collected at Various Locations in the United States 29
4. Dimensions and Number of Septa of Pycnidiospores and
Diameters of Pycnidia of Septoria chrysanthemella
Specimens Collected at Various Locations in the United
States ...... ...... 30
5. Comparison of Pycnidiospore Dimensions on Six Chrysan
themum Varieties Inoculated with Isolate No. 1 of
Septoria obesa 31
6.Comparison of Pycnidiospore Dimensions on Six Chrysan
themum Varieties Inoculated with Isolate No. 1 of
Septoria chrysanthemella ....... 32
7. Comparison of Septoria obesa and Septoria chrysanthe
mella Specimens Collected in the United states:
Pycnidiospores and Pycnidia 34
8. The Effect of Hydrogen-ion Concentration on the Growth
of Septoria obesa in a Synthetic Liquid Medium ... 40
9.The Effect of Hydrogen-ion Concentration on the Growth
of Septoria chrysanthemel1a in a Synthetic Liquid
Medium 41
10.The Effect of Temperature on the Growth of Septoria
obesa in Synthetic Liquid Media 42
11.The Effect of Temperature on the Growth of Septoria
obesa on Semi-synthetic Agar Medium 43
12.The Effect of Temperature on the Growth of Septoria
chrysanthemella in Synthetic Liquid Meditan ..... 44
iv


19
1:1000 mercuric chloride for 2 minutes and then for 5 minutes each in
3 changes of sterile distilled water. The diseased leaves were then
placed on moist filter paper in sterile petri dishes and maintained
for 24 hours in a constant temperature chamber at 8C. At the end of
this period pycnidiospores in gelatinous matrices had oozed from many
of the pycnidia. With the aid of a stereoscopic microscope, spore
suspensions were prepared by touching the spore masses with the tip
of a sterile needle and rinsing the needle tip in sterile, distilled
water. The suspensions were then plated on water agar in petri dishes
and incubated for about 43 hours at 20C. Bacterial contaminants had
formed colonies by the end of this period and were easily avoided.
Germinated spores were removed singly with a hooked needle and trans
ferred to potato-dextrose-agar medium in petri dishes.
Measurements of pycnidiospores obtained from several chrysan
themum varieties previously artificially inoculated with isolate No. 1
of Septoria chrysanthemella or with isolate No. 1 of Septoria obesa
were made by using the procedures described earlier in this section.
The artificially inoculated material was from an experiment concerned
primarily with pathogenicity, and an account of the techniques employed
is given under that topic.
Studies of morphological characters of the fungi in host leaves
and in culture on potato-dextrose-agar medium were made from serial
sections prepared by the paraffin method and examined unstained or
after being stained with safranin, safranin-fast green, cotton blue,
or iron hematoxylin


INTRODUCTION
Two distinct septoria diseases of the cultivated chrysanthemum
commonly occur in the United States and in several other parts of the
world where the host is grown.
The development of similar disease symptoms has led to un
certainty and error. Confusion abounds with respect to the identity
of the causal organism. Information is incomplete or lacking cm the
morphology and physiology of the causal parasites and also on their
relative pathogenicity on various varieties of the cultivated chrysan
themum. Various host-parasite relationships are not known, especially
the mode of penetration by the fungus and pathological anatomy.
These investigations were inaugurated to explore those areas
where information is incomplete or lacking with respect to the
diseases, the pathogens, and the host-pathogen relations.
The results are reported herewith. The experimental work is
supplemented by a review of the literature in all areas pertaining
to the diseases, the pathogens, and the host-pathogen relations.
1


31
TABUS 5
COMPARISON OP PYCNIDIOSPORE DIMENSIONS ON SIX CHRYSANTHEMUM
VARIETIES INOCULATED WITH ISOLAIS HO. 1 OP SBPTORIA OBESA
Chrysanthemum Variety
Length in
Microns
Range
Mean*5
Alaska
49-101
74.5
Gold Ball
48-100
74.1
Illini Regal
46-96
71.7
mini Warpaint
53-102
76.0
Jetfire
50-114
74.0
Mary L. Hall
47-99
72.8
Overall
46-114
73.8
aThirty-four pycnidiospores were measured in each variety.
bThere vas no significant difference in mean length of spores
among varieties at the 5 per cent level*


9
Fig. 2.Lesions on young chrysanthemum leaves 6 weeks
after inoculation with spores of Septoria obesa (X 3/4).


64
TABLE 22
SEVERITY OF DISEASE ON FOURTEEN CHRYSANTHEMUM VARIETIES INOCULATED
WITH SEPTORIA CHRYSANTHEMELLA IN THE FIELD
Chrysanthemum
Variety
No. of No. of Plants in Each Disease Class
Plants Severe Moderate Mild Healthy
Alaska 4
Beauregard 4
Bonnie 4
Dark Buckley 4
Delaware 4
Gold Ball 4
Illini Regal 4
Indianapolis Bronze 4
Jetfire 4
Mary L. Hall 4
Portrait 4
Rayonnante 4
White Top 4
Yellow Shasta 4
Control Plants 14
2
3
1
2
2
2
3
3
1
2
1
1
1
2
2
1
1
2
2
2
1
2
1
1
5
1
3
1
2
1
4
2
1
9


THE CAUSAL ORGANISMS
Taxonomy
The identity of the numerous species of Septoria is based on
morphological differences, physiological adaptations, and specificity
for different host plants. Regardless of morphological similarities,
Septoria spp. specific for different, not-closely-related hosts are
placed in separate species. Morphologically similar septort sis which
are specific for different but closely-related hosts, such as hosts
in the same genus or family, are usually placed in different species
but are sometimes given varietal names in the same species.
In discussing the taxonomy of the two causal organisms under
consideration, septorlas described on different host plants will be
considered to be of different species unless proved identical by
cross-inoculation experiments.
SEPTORIA CHRYSANTHEMELLA SACC.In 1892 Cavara (32) described
a fungus causing spots on chrysanthemum leaves in Italy. He gave
this fungus the binomial Septoria chrysanthemi Cav. However, since
Allescher (32) had used this name in 1891 in describing a very similar
fungus on the leaves of Chrysanthemum leucanthemum in Bavaria, Saccardo
(32), in 1895, changed the name of Cavara*s fungus to Septoria chrysan-
themella Cav. In 1907 Magnus (25) stated that the latter fungus should
12


48
TABLE 15
THE EFFECT OF CARBON SOURCE ON THE GROWTH OF SEPTORIA
CHRYSANTHEf.SELLA ON SEMI-SYNTHETIC AGAR MEDIUM
Carbon
Source'3
Culture Diameter
Range
in mma
Mean0
Sporulatioa
Mannose
32.5-34.5
33.6
Yes
D-mannitol
25.0-27.0
26.0
Yes
Maltose
32.5-33.5
33.2
Yes
Galactose
35.0-37.0
36.1
Yes
Sucrose
35.5-43.5
40.6
Yes
Dextrose
35.5-37.0
36.2
Yes
Lactose
24.0-25.5
24.9
Yes
Minus carbon source
16.0-13.5
17.2
Yes
a
After 23 days of growth.
bAt the rate of 10 g per liter.
^our replications were used.
Septoria obesa cultures were very dark or black on all nitrogen
sources used and also on media lacking nitrogen; however, with glycine
and also with the absence of nitrogen, the cultures were less raised and
much less dense than with the other nitrogen sources. Pycnidiospores
were produced in every culture but were very sparse when glycine was
used and when nitrogen was lacking. The amount of growth of this fungus
on the various nitrogen sources is given in Table 16.