Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations
1986 Epidemiology of Mycosphaerella populorum Thompson on the foliage and stems of Populus species Christopher John Luley Iowa State University
Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Agriculture Commons, and the Plant Pathology Commons
Recommended Citation Luley, Christopher John, "Epidemiology of Mycosphaerella populorum Thompson on the foliage and stems of Populus species " (1986). Retrospective Theses and Dissertations. 8096. https://lib.dr.iastate.edu/rtd/8096
This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. INFORMATION TO USERS
This reproduction was made from a copy ofa manuscript sent to us for publication and microfilming. While the most advanced technology has been used to pho tograph and reproduce this manuscript, the quality of the reproduction is heavily dependent upon the quality of the material submitted. Pages in any manuscript may have indistinct print. In all cases the best available copy has been filmed.
The following explanation oftechniques is provided to help clarify notations which may appear on this reproduction.
1. Manuscripts may not always be complete. When it is not possible to obtain missing pages, a note appears to indicate this.
2. When copyrighted materials are removed from the manuscript, a note ap pears to indicate this.
3. Oversize materials(maps, drawings, and charts)are photographed 1^sec tioning the original, beginning at the upper left hand comer and continu ing from left to right in equal sections with small overlaps. E)ach oversize page is also filmed as one exposure and is available, for an additional charge, as a standard 35mm slide or in black and white paper format.*
4. Most photographs reproduce acceptably on positive microfilm or micro fiche but lack clarity on xerographic copies made from the microfilm. For an additional charge, all photographs are available in black and white standard 35mm slide format.*
*For more information about black and white slides or enlarged paper reproductions, please contact the Dissertations Customer Services Department.
T T-\yf-T Dissertation LJIVIX Information Service
University Microfilms International A Bell & Howell Information Company 300 N. Zeeb Road, Ann Arbor, Michigan 48108
8627131
Luiey, Christopher John
EPIDEMIOLOGY OF MYCOSPHAERELLA POPULORUM THOMPSON ON THE FOLIAGE AND STEMS OF POPULUS SPECIES
Iowa State University PH.D. 1986
University Microfilms
I ntsrn&tion&l 300 N. Zeeb Road. Ann Arbor, Ml48106
PLEASE NOTE:
In all cases this material has been filmed in the best possible way from the available copy. Problems encountered with this document have been identified here with a check mark V .
1. Glossy photographs or pages
2. Colored illustrations, paper or print
3. Photographs with dark background
4. Illustrations are poor copy
5. Pages with black marks, not original copy
6. Print shows through as there is text on both sides of page
7. Indistinct, broken or small print on several pages
8. Print exceeds margin requirements
9. Tightly bound copy with print lost in spine
10. Computer printout pages with indistinct print
11. Page(s) lacking when material received, and not available from school or author.
12. Page(s) seem to be missing in numbering only as text follows.
13. Two pages numbered . Text follows.
14. Curling and wrinkled pages
15. Dissertation contains pages with print at a slant, filmed as received
16. Other
University Microfilms International
Epidemiology of Mycosphaerella populorum Thompson on the foliage and
stems of Populus species
by
Christopher John Luley
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department: Plant Pathology, Seed and Weed Sciences Major; Plant Pathology
Approved:
Signature was redacted for privacy. xri èharge of
Signature was redacted for privacy. For the Majgp/Department
Signature was redacted for privacy.
Iowa State University Ames, Iowa
1986 ii
TABLE OF CONTENTS
Page
GENERAL INTRODUCTION 1
SECTION I. ASCOSPORE PRODUCTION, RELEASE AND INFECTION 5 OF POPULUS BY MYCOSPHAERELLA POPULORUM
INTRODUCTION 6
LITERATURE REVIEW 8
MATERIALS AND METHODS 20
RESULTS 29
DISCUSSION 57
LITERATURE CITED 65
SECTION II. CONIDIAL RELEASE, GERMINATION AND INFECTION 71 OF POPULUS BY SEPTORIA MUSIVA
INTRODUCTION 72
LITERATURE REVIEW 74
MATERIALS AND METHODS 84
RESULTS 87
DISCUSSION 111
LITERATURE CITED 117
SECTION III. IN VITRO ASCOCARP PRODUCTION 121 OF MYCOSPHAERELLA POPULORUM
INTRODUCTION 122
LITERATURE REVIEW 123
MATERIALS AND METHODS 129
RESULTS 131
DISCUSSION 134 iii
Page
LITERATURE CITED 135
SUMMARY AND CONCLUSIONS 138
LITERATURE CITED 143
APPENDIX: FIGURES AND TABLES 145
ACKNOWLEDGMENTS 157 1
GENERAL INTRODUCTION
Septoria leaf spot and canker are serious foliar and stem diseases of certain Populus species and hybrids in North America (Waterman,
1954). Introduced poplars or hybrids with P. trichocarpa Torr. and
Gray, 2i, balsamifera L., or deltoides Bartr. parentage were reported as being particularly susceptible (Waterman, 1954). Resistance to both the leaf spot and canker can vary greatly among locations and hybrids of common parentage (Ostry and McNabb, 1985; Cooper and Filer, 1976). Most literature attributes foliar and stem infection to the anamorph,
Septoria musiva Peck, which is commonly isolated from stems and leaves during the growing season (Bier, 1939). Thompson's (1941) description of the loculoascomycete Mycosphaerella populorum Thompson identified the perfect state of the fungus and established overwintered leaves as the source of primary inoculum in the spring. Additional research is still needed to determine the relative contribution of each spore state to intensification of the leaf and stem diseases in the field.
The impact of Septoria leaf spot and canker in natural forest ecosystems is minimal although all indigenous Populus species tested were found susceptible to foliar infection (Waterman, 1954). Balances inherent in forest communities (Dinus, 1974) apparently limit M. populorum to an endemic leaf spot pathogen. Selection of Populus and
Populus hybrids for utilization in intensive culture systems has dramatically increased the importance of both the foliar and stem disease. 2
Selection for resistance to diseases has been a priority since the
initiation of Populus breeding programs for "mini-rotation" or short
rotation forestry (Schreiner, 1970; Stout and Schreiner, 1933). Despite
this, damage caused by infection of foliage and stems by ^ populorum
has limited use of many fast growing Populus hybrids in short rotation,
intensive culture (Waterman, 1954; Ostry and McNabb, 1985). Disease
losses have continued to occur in nurseries and stool beds (Zalasky,
1978; Filer et al., 1971), shelterbelts and plantations (Bier, 1939;
Waterman, 1954; Weiss et al., 1976) and stands coppiced for regeneration
(Moore et al., 1982; McNabb et al., 1982). In experimental plantations
in Iowa, populorum reduced biomass production of a susceptible clone
to 632 of a resistant clone after three years growth (McNabb et al.,
1932). Stand reduction from stem infection and girdling was 30Ï one
year after coppicing on the same clone. Similar, although unquantified,
reports of losses have been made from Canada (Bier, 1939; Zalasky et
al., 1968) and northeastern (Waterman, 1954) and southern (Weiss et al.,
1976) United States.
Despite the apparent omnipresence and importance of populorum to
intensive Populus production, the epidemiology of populorum has been
virtually unstudied. Quantitative analysis of epidemics in other forest
and agricultural pathosystems has facilitated an understanding of the factors that contribute to disease outbreaks and has set the strategy for the development of management practices (Van Der Plank, 1963). Most quantitative epidemiological research on 2^ populorum has focused on either studying the effect of silvicultural practices on disease losses 3
(Bowersox and Merrill, 1976; Moore et al., 1982; McNabb et al., 1982) or
on identifying resistance present in fast growing Populus selections
(Ostry and McNabb, 1985; Cooper and Filer, 1976). These studies have
contributed important observations on the various host, pathogen and
environmental factors that appeared to affect disease development.
Factors that have been observed to affect disease buildup include microclimatic conditions associated with stand density (Waterman, 1954;
McNabb et al., 1932), inoculum density and source (Waterman, 1954;
Waterman and Aldrich, 1952; McNabb et al., 1982), availability of infection courts (Bowersox and Merrill, 1976) and host resistance (Ostry and McNabb,1985; Waterman, 1954; Bier 1939). Gerstenberger (1983) formalized these observations with a conceptual model of the
Mycosphaerella-Populus system. In most cases, the host, pathogen and environmental factors were not quantified or studied further to determine how they affected the epidemiology of 2^ populorum.
Mathematical modeling and simulation of epidemics can help to reveal underlying relationships and are prerequisites to developing disease forecasting systems (Kranz, 1974). Quantification and mathematical modeling in a number of other tree disease systems, such as white pine blister rust (McDonald et al., 1981) and apple scab (Jones,
1978) have allowed prediction of the conditions which lead to disease outbreaks. This approach should be applicable and equally beneficial in the Mycosphaerella-Populus pathosystem. Therefore, the main objective of this study was to analyze quantitatively the epidemiology of M. populorum under controlled and field conditions. The specific 4
objectives and results of the study will be presented in three sections.
The first section will focus on ascospore production, release and infection of two Populus clones by populorum. The second section will deal primarily with field and laboratory studies on the infection of stems and leaves of the same two clones by conidia of ^ musiva. The final section is a closely related study on the ^ vitro production of ascocarps of populorum. 5
SECTION I. ASCOSPORE PRODUCTION, RELEASE AND INFECTION OF POPDLUS BY MYCOSPHAERELLA POPDLORDM 6
INTRODUCTION
The source of primary inoculum and the role of primary inoculum in
contributing to epidemics have been the subjects of many intensive
studies. Primary inoculum of temperate zone foliar pathogens is typically an ascigerous overwintering state that develops in leaves or other infected plant parts by the spring of the year. This sexual state is significant in the epidmiology of foliar diseases because it is the link bridging the parasitic phases of the fungus and is a source of genetic recombination in the pathogen population. However, identification of the sexual overwintering state and determination of its role in epidemics frequently lags far behind the study of the asexual states that perpetuate the pathogen during the growing season
(Zadoks and Schein, 1979).
Identification of the ascigerous overwintering fungus
Mycosphaerella populorum occurred more than 55 years after Peck (1884) identified Septoria musiva as the cause of a leaf spot on Populus deltoides Bartr. The role of populorum in intensification of the foliar and stem disease still remains unclear. Observations have suggested that ascospores produced in pseudothecia in overwintered leaves are the main source of primary infection (Bier, 1939; Waterman,
1954) and that ascospores can infect both stems and leaves (Filer et al., 1971). No published studies have quantified ascospore production or related ascospore release to infection of foliage and stems.
Intensive study of the ascigerous state of pathogens with disease 7
cycles similar to Septoria leaf spot and canker has been valuable in
formulating their disease control measures. Particularly, over 80 years
of intensive study on the apple scab pathogen, Venturia inaequalis
(Cke.) Wint., directly resulted in a number of predictive and
forecasting programs (Jones, 1978), more efficacious use of fungicides
(Gilpatrik, 1978), and alternative methods of managing primary inoculum
(Ross and Newberry, 1975). Extensive research on ascospore production and release and on conditions necessary for infection by inaequalis also provide an excellent model for similar studies on populorum.
The objective of this study was to evaluate the role of primary inoculum produced by populorum in foliar and stem disease intensification. Specifically, the objectives were to (1) quantify ascospore production and release throughout the growing season (2) determine and model the effects of the environment on ascospore release and (3) quantify ascospore infection of stems and leaves of two Populus clones within a Mycosphaerella infected poplar plantation. 8
LITERATURE REVIEW
Mycosphaerella populorum is a widely distributed loculoascomycete
associated with the fallen leaves of Populus species in North America
(Thompson, 1941; Barr, 1972). All indigenous Populus species tested
were found to be susceptible to foliar infection (Waterman, 1954; Bier,
1939) and accordingly populorum has been reported from most areas in
North America where poplars are found. The sole report of the fungus
outside North America was made by Sarasola (1944) who identified
Septoria musiva as the cause of cankers on Populus in Argentina,
Thompson's (1941) description of M. populorum completed the disease
cycle of Septoria leaf spot and canker. The cycle is similar to many
other temperate ascomycete foliar pathogens (Manion, 1981). Review
relative to the epidemiology of the pathogen and the importance of the
ascigerous state in the disease cycle is limited, because relatively few
studies have been conducted on the topic. Comparisons with a
taxonomically and epidemiologically similar ascomycete, Venturia
inaequalis (Cke.) Wint., and the overwintering ascogenous states of a
number of other foliar pathogens will be made to supplement the
discussion.
Sources of Primary Inoculum
The main source of primary inoculum of populorum is believed to be ascocarps that develop in overwintered leaves. Large numbers of mature pseudothecia with ascospores have been found in fallen leaves in 9
the spring (Waterman, 1954; Bier, 1939). Bier (1939) and Waterman
(1954) also reported finding ascocarps in infected branch tissue and in
leaves persisting on branches that had died the previous spring.
Ascocarps in infected branches are probably an insignificant source of
primary inoculum because the fungus is quickly displaced from stem
tissue by other fungi (Waterman, 1954).
Overwintering of Ascocarps
Ascocarps of ^ populorum develop to maturity in fallen leaves in
the spring of the year (Thompson, 1941) although the state in which the
fungus overwinters is not known. Production of spermagonia in the fall
(Thompson, 1941) suggested that the early ascocarp developmental stages
transpired at this time. Higgins (1936) found that the initial stages
of ascocarp development of M. tuliferae (Schw.) Higgins occur when
spermagonia appear in the fall. Pseudothecia with archicarps
(ascogonia) were found between the first of October and the middle of
December. Further ascogonial development did not occur until the latter
part of January and young asci were found in February. Similar observations were made on M. personata Hig. (Higgins, 1929), M. arachidicola Jenk and ^ berkelyii Jenk. (Jenkins, 1938) on their respective hosts.
Wilson (1928) conducted one of the first in-depth studies on overwintering stages of inaequalis and on environmental effects on initiation and development of pseudothecia. This study and a subsequent report (Gadoury and MacHardy, 1982a) have shown most ascocarps are 10
initiated within a month after leaf fall and are directly associated
with lesions that developed during the growing season. James and Sutton
(1982a) observed that ascogonial initials and pseudoparaphyses developed
in the lumen of pseudothecia in the fall. An inherent dormant period of
approximately 45 days followed these initial stages of pseudothecial
development and prohibited the further maturation of asci and ascocarps.
Temperature and moisture relationships also directly affected
pseudothecial initiation and maturation (Wilson, 1928). Pseudothecial
initiation required moisture (Wilson, 1928); a lack of moisture delayed
but did not inhibit further pseudothecial development (Wilson, 1928;
James and Sutton, 1982a,b). Temperature effects were mostly
quantitative and directly affected the rate of pseudothecial initiation
(Wilson, 1928), number of pseudothecia per unit area (Wilson, 1928; Ross
and Hamlin, 1962; O'Leary and Sutton, 1986), rate of ascospore
maturation (James and Sutton, 1982a; Gadoury and MacHardy, 1982a,b), size of pseudothecia and number of asci per pseudothecium (Gadoury and
MacHardy, 1982a). Temperature effects were manifested primarily when moisture was not limiting (James and Sutton, 1982a). Other factors such as time of leaf fall (Wilson, 1928; Gadoury and MacHardy, 1982a) and lesion type (Wilson, 1928) also affected ascocarp initiation and development.
Studies on pseudothecial initiation and on environmental influences on pseudothecial development have been useful in the creation and evaluation of models predicting ascospore maturity. For example, knowledge of the inherent dormant period of ascocarps in the fall was 11
used to explain large deviations in an ascospore maturity model when
used in North Carolina. The model closely predicted ascospore
maturation in New York where it was developed and where ascospore
development was not affected by the dormant period (Sutton et al.,
1981). A relationship between pseudothecial initiation and foliar
lesions apparent during the growing season has permitted forecasting
ascospore levels in commercial apple orchards (Lewis, 1980; Gadoury and
MacHardy, 1986). Study of pseudothecial ontogeny has also allowed more
precise evaluation of the effect of fungicides that inhibit
pseudothecial initiation and development (Ross and Newberry, 1975).
Niyo et al. (1986) studied the development of ascocarps of M.
populorum from leaves collected in December and maintained in a cold
room. She found ascocarp development of populorum was typical of
that described for the Dothideales (Luttrell, 1973). However, stages of
ascocarp development were not related to host phenology or environmental
factors.
Maturation of Asci and Ascospores
Bier (1939) stated that ascospore discharge commences with Populus
bud opening but did not indicate the species involved or the source of his observation on ascospore discharge. Thompson (1941) collected ascospores on May 1 after exposure of glass slides three days earlier to overwintered poplar leaves. Ascus and ascospore maturation apparently occurs in the early spring. However, relation to host phenology and the effect of the environment on the maturation process remain to be 12
described for 2^ populorum.
The relationships among ascospore maturity, host phenology and the
environment are of critical importance in the control of foliar
diseases. Fungicide sprays to protect foliage and other susceptible
plant parts from primary infection should be applied concurrently with
initial ascospore release. Extensive study focused on ascospore
maturation of Drepanopeziza punctiformis Gremm. (the cause of Marssonina
anthracnose of poplars) showed ascospores are mature by the time leaves
flush (de Kam, 1975). Fungicide sprays to control this disease in the
Netherlands are now based on the timing of poplar leaf flush in the
spring. More extensive study on ascospore maturation has been conducted
for V. inaequalis in the United States. Szkolnik (1969) and MacHardy
and Gadoury (1985) have consistently found ascospore maturation of V.
inaequalis coincides with the early stages of flower bud expansion in
most years. At least 1% of the ascospores of this fungus are mature by
the time of silver tip phenophase on the variety Mcintosh (MacHardy and
Gadoury, 1985). Occasional large deviations have been noted in some
years and are often related to abnormal winter weather (Gilpatrick and
Szkolnik, 1978). Massie and Szkolnik (1974) predicted ascospore
maturity with a regression model using degree day accumulation and
precipitation measurements from leaf fall. James and Sutton (1982b)
used temperature measurements from February first to predict the developmental stages of ascocarps and to estimate the timing of ascospore maturity. They found that pseudothecium maturation was directly related to temperature when moisture is not limiting. Daily 13
moisture levels below 0.25 mm rain or less than 12 hours of 100?
relative humidity was determined sufficient to delay ascocarp
maturation. MacHardy and Gadoury (1985), however, found that such
moisture limitations did not affect ascospore maturation in New
Hampshire. Significant deviations from these models are not uncommon
when extremes in moisture availability occur or when they are used
outside the region in which they were developed (Sutton et al., 1981;
James and Sutton, 1982b). The models, although not fully confirmed in
practice (Gilpatrick and Szkolnik, 1978), provide a basis for further
study of temperature and moisture relations and other factors that
affect ascocarp maturation.
Duration and Seasonal Pattern of Ascospore Production
Ascospore production and discharge by ^ populorum apparently continues intermittently throughout much of the growing season.
Thompson (1941) collected viable ascospores from the first of May until the end of August in New York during an unusually dry spring and summer.
Gerstenberger (1984) found that ascospore discharge in Michigan continued throughout much of the' growing season and collected spores until the end of August. These pilot studies suggest that ascospores of
M. populorum are produced over an extended time period during the growing season. Pseudothecia of pinodes (Berk, and Blox.) Vesterg. produced ascospores for over 75 weeks when maintained in laboratory experiments (Carter, 1963). In Wisconsin, Hare and Walker (1944) found that ascospores of the same fungus could be discharged over 18 months in 14
the field. In comparison, pseudothecia of inaequalis have a relatively short productive period, spanning about two to three months
(MacHardy and Gadoury, 1985; Hirst and Stedman, 1961) or about 30 wetting periods from rain (Hirst and Stedman, 1961).
Ascospore productivity, or the number of ascospores produced per cm^ of dead leaf tissue (Hirst and Stedman, 1962), of many temperate ascomycetes shows a distinct seasonal pattern (McGee and Petrie, 1979;
Ingold, 1961). Seasonal production of ascospores of V. inaequalis exhibits a distinct progression from mostly immature-to-mature-to- degeneration of spores and asci (Gilpatrick and Szkolnik, 1978).
MacHardy and Gadoury (1985) identified this as three phases in development of ascospore populations. The first was a lag phase (0-10% maturity), followed by an accelerated phase (11-90% maturity), and a final phase (91-100% maturity). They also quantified a direct relationship between the cumulative population of mature ascospores and degree day accumulation from the time ascospores first matured in the spring (Gadoury and MacHardy, 1982b). The model has been useful in determining the rate of ascospore discharge and the length of primary infection period in the Northeast. The model uses silver tip phenophase on Mcintosh apple, or the estimated time of first ascospore maturation of V. inaequalis (MacHardy and Gadoury, 1985), as a biofix to initiate the prediction of spore maturity. Historic degree day data can then be used to predict the length of primary infection periods from the time ascospores first mature. Prediction of ascospore maturity by the model has been implemented as a tool to aid apple growers in New England 15
(MacHardy and Gadoury, 1985). Research on M. populorum may allow
development of a similar predicitve system.
Environmental Effects on Ascospore Release
Thompson (1941) and Gerstenberger (1983) both reported the release
of ascospores of 2^ populorum after wetting of leaves by rain. Rainfall
is considered essential for release of epidemiologically significant
numbers of ascospores of inaequalis (Hirst and Stedman, 1962; Brook,
1969b). Dew releases relatively few ascospores of the apple scab fungus
in comparison to wettings by rain (Hirst and Stedman, 1962; Brook,
1969b). Leaf wettings from dew, however, are important in the release
of ascospores of pinoides (Carter, 1963)» fijiensis Meredith
(Meredith et al., 1973) and other ascomycetes (Hirst, 1953).
A daily periodicity of spore release by ^ inaequalis was noted by
Hirst and Stedman (1962). More spores were present in the air during
the middle of the day than at any other time. They could not attribute
this periodicity to the individual affect of light, temperature or
relative humidity alone. Brook (1969a) later suggested the periodicity
was due to the affect of light on ascospore release. Few ascospores are
released during the night even if wetted by rain because darkness
inhibits ascospore discharge (Brook, 1969a). In the same study, far red
light was found to stimulate spore release by inaequalis while
infrared light suppressed release. Diurnal periodicity of ascospore
release has also been documented in M. pinoides (Carter, 1963), M. fijiensis (Meredith et al., 1973), M. citricola Whiteside (Whiteside, 16
1970) and ^ musicola Leach (Meredith, 1962), although the exact
mechanisms controlling the periodicity have not been clearly defined.
Role of Ascospores in Disease Intensification
Infection of stems and leaves early in the spring has been related
to the flux of ascospores from leaf litter (Waterman, 1954). Leaf spot
symptoms first appear three to four weeks after leaves begin to first
emerge and ascospores are purportedly released (Bier, 1939). Foliar
symptoms are most severe and develop first on lower branches (Bier,
1939; Waterman, 1954). Waterman (1954) attributed a high percentage of
this early spring infection to ascospores of 2^ populorum released from
overwintered leaves.
The role of ascospores of 2^ populorum in stem infection is less
clear. Filer et al. (1971) exposed stems of a susceptible P. deltoides
clone to overwintered leaves with 2% populorum fruiting bodies. The
stems and leaves were placed in a mist chamber for a seven day period
and then were removed to a greenhouse to allow symptoms to develop.
Sixty percent of the stems developed stem cankers which were attributed to M. populorum. McNabb et al. (1982) suggested that multiple infection of stems in a dense coppiced stand resulted from the accumulation of leaf litter around stems. Ascospore or conidial inoculum levels were not reported; however, high infection levels may have been partially due to high ascospore levels around stems in the spring (McNabb et al.,
1982).
Ascosporic inoculum is requisite for epidemics of apple scab 17
(Lewis, 1980). Decreased inoculum levels of V. inaequalis with the
advent of better fungicide control during the growing season has
resulted in decreased levels of primary infection in the Northeast
(Lewis, 1980). Similarly, the amount and severity of infection of
rapeseed by Leptosphaeria maculans (Desm.) Ces. et de Not. has been
related to levels of ascospore inoculum present during periods of crop
susceptibilty (McGee and Petrie, 1979). Incidence of Septoria leaf
blotch of wheat has also been related to the proximity of wheat fields
to overwintering inoculum of graminicola (Fuckel) Schr. and
Leptosphaeria nodorum Muller (Sanderson et al., 1985).
Management of Primary Inoculum
Management of sources of primary inoculum and control of primary infection are the foundation for control of apple scab (Lewis, 1980) and numerous other diseases (Van Der Plank, 1963; Berger, 1977). Reduction in the amount and time of primary infection delays the onset and severity of epidemics (Van Der Plank, 1963). Management of sources of primary inoculum of populorum may be an important part of integrated control of this pathogen (McNabb'et al., 1982).
Several strategies of primary inoculum management may be potentially useful. Removal of the source of primary inoculum in the spring with various cultural practices such as raking or cultivation may be feasible and could significantly reduce overwintering populations of
M. populorum (McNabb et al., 1982). Deep plowing of crop residues and crop rotation to reduce sources of primary inoculum are common and 18
effective means of disease control of many field crop pathogens (Berger,
1977; Nyvall, 1979). Levels of reduction of overwintering populations
of H. populorum may be particularly important and require investigation.
Utilization of pathogen free planting stock should be considered a
priority when establishing new plantations (Ostry and McNabb, 1982).
Conidia of ^ musiva can survive in lenticels and in resinous coatings on uninfected buds (Waterman and Aldrich, 1952; Ford and Waterman,
1954). Surface sterilization (Waterman and Aldrich, 1952; Ford and
Waterman, 1954) or fungicide dips of dormant cuttings (Ostry and McNabb,
1982) significantly reduced chances of using diseased planting stock.
Natural Populus species are a potential reservoir of inoculum for
M. populorum (Bier, 1939; Waterman, 1954; Thompsom, 1941). Isolates infecting only the leaves of native poplars can infect both leaves and stems of susceptible hybrids (Zalasky, 1978; Waterman, 1954; Bier,
1939). Locating plantations an adequate distance from natural sources of inoculum may delay the onset of epidemics sufficiently to minimize losses in short rotations. The distances required to avoid infection early in stand rotation remain to be determined.
Fungicides may have a place in the control of Populus diseases in short rotation, intensive culture systems if the value of wood produced justifies their use. A number of fungicides can effectively control M. populorum with as few as three spray treatments per season (McNabb et al., 1982; Carlson, 1972, 1974). Research on proper timing of applications, targeted at the control of primary infection, may show that a limited number of applications or single application techniques 19
CGilpatrick, 1978) could control both stem and foliar infection.
The current low value of wood produced in poplar plantations will limit the usefulness of control measures that require substantial capital input. Utilization of host resistance offers the most economically and biologically attractive long term method of disease control. Sufficient host resistance has been identified in silviculturally acceptable Populus selections to warrant their use in intensive culture (Ostry and McNabb, 1985). However, host resistance can vary greatly among locations (Ostry and McNabb, 1985; Cooper and
Filer, 1976), making it necessary to screen selections locally before large scale planting is implemented. The long term nature of forest crops makes them particularly vulnerable to virulence changes in pathogen populations. Research on the teleomorph of this pathogen is warranted because shifts in pathogen populations towards increased virulence will be mediated largely through the sexual cycle. 20
MATERIALS AND METHODS
Field Experiments and General Sampling Design
Field trials were conducted within a mixed hybrid Populus
plantation planted in 1977 at the Iowa Conservation Commission nursery
in Ames, Iowa. The plantation was originally established for pest resistance evaluation of Populus clones with varied parentage (Ostry and
McNabb, 1985). Three sites were selected within the plantation in proximity to Mycosphaerella populorum susceptible clones. Five rooted
potted cuttings of clone NC5272, 3 rooted potted cuttings of clone
NC5271t 2 microscope slides with the trapping surface facing up 30 cm from ground level, 2 vaseline coated microscope slides with the trapping surface facing down 10 cm from ground level, two monofilament spore traps and 1 or 2 nylon mesh bags containing samples for ascospore liberation tunnel experiments were located at each site. Weekly sampling periods ran from Julian date (JD) 107 to JD 274 in 1984 and JD
92 to 326 in 1985. Exposure of potted cuttings was started JD 107 and discontinued on JD 276 in 1985 due to frost. Julian dates designated in this study refer to days of the year (Seem and Eisensmith, 1986).
Environmental data
Located in the center sampling station was a Campbell Scientific
CR21 micrologger (Campbell Scientific, Logan, Utah 84321). The unit stored hourly average temperature and relative humidity and maximum relative humidity sensed by a Phys-Chemical Research Model PCRC011 RH 21
sensor and a Fenwal UUT-51j1 thermistor (Phys-Chemical Res. Corp., New
York, New York 10011). Leaf wetness was recorded as a proportion of each hour that moisture was sensed on a circuit board of interlacing fingers of gold plated copper. A layer of flat latex paint (Gillespie and Kidd, 1978) spread water droplets across the surface of the plate.
A seven-day recording hygrothermograph also was maintained at the site.
Rainfall data were obtained from the NOAA registered weather service station at the Ames Pollution Control Plant located approximately 0.5 miles north of the plantation. Temperature data for degree day calculations and the development of a degree day model from
30 years (1951-1980) of climatic data was obtained from a U.S. Weather
Bureau station located 8 miles WSW of Ames. Degree days were calculated using the rectangular method (maximum daily temperature+minimum daily temperature/2) (Arnold, I960).
Stem and foliar infection periods
Disease-free rooted potted cuttings from Populus clones NC5271 and
NC5272 were exposed at the same sites spore traps were located. The pots were sunk 10 cm in the ground and were watered as needed during the week. Clone NC5271 (NE19), a Populus nigra var. charkowiensis X P. nigra var. caudina cross, was reported to be moderately resistant to foliar and stem infection by M. populorum. NC5272 (NED is a (P. nigra
X P^ laurifolia) 'Strathglass' cross and was found to be highly susceptible to foliar and stem infection (Ostry and McNabb, 1985). Both clones were rooted under intermittent mist 4 to 6 weeks before exposure 22
in the field and potted in 10 cm pots in a 1:1:1
peat:perlite:vermiculite mix. The cuttings were fertilized initially
with 7-40-6 magnesium-ammonium nitrate-phosphorus slow release
fertilizer at a rate of 2.95 kg per .17 cubic liter and twice weekly
with 20-19-18 nitrogen-phosphorus-potassium in solution with 5 ppm
FeEDTA. The cuttings were sprayed weekly with the Pencapp M (Zoran,
Co.) for spider mite control. After 7 days exposure in the field,
plants were returned to the greenhouse to allow symptom development and
disease evaluations. Foliar infection was rated 18 days after exposure
on a 0-5 scale where 0 = no infection, 1 = 1-10, 2 = 11-25, 3 = 26-40, 4
= 41-75 and 5 = 75+ lesions per leaf. The first five leaves of clone
NC5272 exhibiting symptoms were evaluated. Clone NC5271 was not
evaluated for foliar infection. Stem infection levels were determined
25 days after exposure in the field. The number of lesions per stem and percentage stems infected were recorded. Rooted potted cuttings maintained in an open area between the greenhouses served as controls.
Spore trap arrangement and data collection
Vaseline spore traps were prepared and used in the field as described by Ostry and Nicholls (1982). Spore densities (spores per cm^) were determined by counts made on 5 transects across the trapping surface at 400X magnification. Ascospores in the larger size range indicated by Barr (1972) (Table 1) were counted. Conidia counts were made in the same manner and conidia were identified as in Palmer et al.
(1980). Table 1. Mycosphaerella species reported from Populus leaves in North America
Species Common host Ascospore measurements Comments
M. populorum Thomp. P. balsamifera L. 16-28 X 4.5-6wm Barr (1972) indicated two (type) 9-21 X 2.5-5ym . ascospore size ranges. 12-28 X 3.5-6ym^ Larger sizes were included in description.
M, populicola Thomp. P_^ tacamahaca Mill 22-32 X 6-6.5pm* (type) a M. orbicularis P.. grandidentata 11-14 X 2-3ym Ascospores colored. Michx, (type) 10-13ym°
M. maculiformis P. alba L. 9-14iim" Common on a range of (Pers. ex. Fr.) P. nigra L. 9-11 X 3-W deciduous tree species. Schrt,
M. macularis P. tremuloides 11-15 X 3-5.5pm^ Spores yellowish-green. (Fr.) Schrt. Michx.
M. populifolia P. balsamifera L. 16-18 X 3.5-W No fruit in type. (Cke.) House P. angulata Ait. Paraphysoids present and no spores in type.'
^Thompson (1941). °Barr (1972). •Ellis and Everhart (1892). Dennis (1968). Table 1. (Continued)
Species Common host Ascospore measurements Comments
H. populnea P. balsamifera L. 11-12 x 2iim® Septorla popullcola^'^ (Sacc.) Hse. (type) conidia In type No ascospores in type.^
^Saccardo (1915). 25
An ascospore liberation tunnel (ALT) was built from plexiglass
according to the specifications of Hirst and Stedman (1962). The ALT
was used to compare the weekly ascospore productivity of a standard area
of overwintered diseased tissue (Hirst and Stedman, 1962; McGee, 1977).
In field experiments, 5 sets of 48, 19 mm diameter leaf discs were cut
from a random selection of overwintered Populus leaves before spores
matured in April. Only leaves with viable ascocarps were used. Leaf
discs held in nylon mesh bags (16 mm^ holes) in the plantation were
returned to the laboratory each week to collect ascospores in the
ascospore liberation tunnel (Fig. 23). The procedure described by Hirst
and Stedman (1962) was utilized, with the following exceptions. If rain
fell in the 24 hours prior to collection the bags with leaf discs were
incubated in a moist chamber overnight. Leaf discs were soaked 3-5
minutes in tap water before wetting with an atomizer from the pressure
side of the vacuum pump. Ascospores were impacted on vaseline coated
slides with an air flow rate of 12 1 per minute for a 2-hour period
before rewetting and collecting for another hour. Each slide was
stained with lactophenol in cotton blue. Spore densities were estimated
by recording the number of spores viewed in 3 fields at 400X and
touching the vertical hash marks of a Leitz 10X eyepiece micrometer. In
1985, a second sample of leaf discs was cut when ascospores first
matured in the field. The discs were placed in opened plastic petri
plates within plastic crispers lined on top and bottom with 2 layers of single-fold moistened paper towels. The crispers were placed in 9, 16, or 21 C temperature chambers. Ascospores were collected every two days 26
during the maximum discharge period and periodically thereafter by-
running the ALT as above except that spores were collected for a one
hour period before rewetting and running an additional hour.
Hourly ascospore concentrations were monitored in 1985 with a
Burkhard seven-day recording spore trap (Burkhard Manufacturing Co. LTD,
Rickmansworth, Hertfordshire, UK). The spore trap was located on the
south end of the plantation juxtaposed to a sampling site. Hourly spore
counts were made as in Hirst (1952) and were compared to hourly
environmental data recorded by the CR21 micrologger.
A monofilament spore trap (Luley and Manion, 1984) was used to trap
conidxa dispersed in water. Conidia were counted as in Luley and Manion
(1984) except a Fuchs Rosenthal hemacytometer was used to determine
spore concentrations after centrifugation. The number of spores per trap was recorded.
Laboratory Experiments
Ascospore germination
Overwintered leaves containing 2^ populorum ascocarps were collected and stored at 8 C until used. Ascospores were discharged onto
1.5% water agar from leaf pieces (16 cm^) adhered to the tops of petri plates. Ascospores were discharged for 15 minutes at room temperature near a window before removing the petri plates and placing them in crispers in 5, 10, 15, 20, 25, 30 or 35 C temperature chambers. Petri plates with germinating spores were removed at 4, 8 and 16 hours. The spores were stained with lactophenol and cotton blue and evaluated for 27
germination, germ tube elongation, and number of germ tubes per spore.
The experiment was replicated over time and 125 spores were measured in
each of the five replicates.
Time analysis of ascospore release
Leaves that were collected and stored at 8 C were used to determine
the rate and duration of ascospore release after a single wetting.
Ascospores were collected during daylight hours from 48, 19 mm diameter
leaf discs by removing a vaseline coated slide at time intervals of 15,
30, 45, 60, 90, 120, 180, 240, 270, 300, and 360 minutes. A clean
vaseline coated slide was inserted at each of the time intervals without
stopping the ascospore liberation tunnel. The leaf discs were initially wetted with 20 ml of tap water and were not wetted thereafter. The experiment was replicted three times.
Data Analysis
The SAS GLM and STEPWISE (SAS Institute, Raleigh, N.C.) procedures were used to relate foliar and stem disease to inoculum and environmental variables measured in the field. The MAXR option, which selects the best model for each variable combination, was specifically used to develop regression equations.
A Gompit transformation where Gompit = -ln(-ln(y)) (where y = proportion of ascospores produced) was used to linearize the plots of cumulative ascospore productivity versus cumulative degree days. The 28
Gompertz transformation is derived from an equation proposed by B.
Gompertz to fit asymmetrical growth curves. The model is particularly
useful in the analysis of growth curves skewed to the right in comparison to a normal logistic growth curve (Berger, 1981). The model has been used in the analysis of biological systems and disease progress curves for a number of pathosystems (Berger, 1981; Chinchilla-Lopez,
1985). Slopes of regression lines or k values (Berger, 1981) are an estimate of the rates of ascospore productivity. Slope values were used to compare differences in controlled temperature data and field data using a t-test. 29
RESULTS
Field and Laboratory Experiments
Ascospore production
Ascospores of Mycosphaerella populorum were first collected with
the ascospore tunnel on JD 111 in 1984, JD 107 in 1985 and JD 93 in
1986. Although sampling was initiated prior in the season, ascospores
were not collected before the 1985 and 1986 collection dates. The time
ascospores matured in 1984 was not determined since spores were
collected the first sampling date (JD 111). Ascospore maturity
corresponds approximately with vegetative bud swell on Populus deltoides
in central Iowa (Table 2).
Ascospore production peaked in late-May in 1984 and mid-May in 1985
(Fig. 1 and 2). Spores were collected by both spore trap methods over an extended time period in the two growing seasons (Fig. 1 and 2).
Spore production declined to extinction by JD 218 in 1984 and JD 240 in
1985 as monitored by the ascospore tunnel. Differences in spore production peaks and duration into the season were evident between the vaseline spore trap and the ascospore liberation tunnel (Fig. 1 and 2).
Rates of tissue degradation of leaf discs used in the ascospore tunnel differed in the two field seasons (Fig. 22).
In controlled temperature chambers, the rate and duration of ascospore production was related to incubation temperature (Fig. 3A).
Ascospore production continued for 74, 98 and 192 days at 21, 16, and 9
C, respectively (Fig. 3A). A sigmoid relationship between ascospore 30
Table 2. Cottonwood phenology and Julian date of initial ascospore collection of Mycosphaerella populorum
Year
1984 1985 1986
Cottonwood flowering^ ND^ 103 90
Bud swell^ 105 106 97
Ascospores collected^ 111® 107 93
^Male flowers fully expanded.
^Not determined.
°Outer bud scales breaking away.
^Collected with the ascospore tunnel.
^Ascospores were collected the first sampling date; therefore, the time of first ascospore maturity was unknown. Figure 1. Rainfall and ascospore densities monitored for 7-day periods during 1984 within a mixed hybrid Populus plantation. Ascospore densities are an average of five samples from the ascospore liberation tunnel and four slides at each of three sites for thCgVasellne spore trap. Vaseline spore trap data are expressed as spores per cm and ascospore tunnel data are the number of spores touching the vertical hash marks in three fields of an eyepiece micrometer and averaged for the five samples 16 Rainfall 12 8 4 0
150
125 1984
100 • flSCOSPORE TUNNEL 75
50
25 0
125 150 175 200 JULIAN DATE Figure 2. Rainfall and ascospore densities monitored for 7-day periods during 1985 within a mixed hybrid Populus plantation. Ascospore densities are an average of five samples from the ascospore liberation tunnel and four slides at each of three sites for thcgvaseline spore trap. Vaseline spore trap data are expressed as spores per cm and ascospore tunnel data are the number of spores touching the vertical hash marks in three fields of an eyepiece micrometer and averaged for the five samples
I 12 10 Rainfall 8 6 4 2 0
350
300
250 1985 200
150 • flSCOSPORE TUNNEL 100 o VASELINE SPORE TRAP
50 0
1 125 150 175 200 225 JULIAN DATE -- - • — 100 A mucosa
80 - Am • 9C 60 - g O 16C A 21 C
40 -
20
50 100 150 400 800 1200 1600 2000 DAYS INCUBATION DEGREE DAYS BASE 0 C
Figure 3A. Cumulative ascospore productivity as determined by the ascospore liberation tunnel In temperature cabinets
Figure 3B. The same cumulative ascospore productivity data plotted against degree days base 0 C 36
productivity and degree day accumulation, base 0 C, was evident from
both controlled temperature (Fig. 3B) and field studies (Fig. 4A and
4B). In 1985, cumulative ascospore production apparently was affected
by dry weather in May and June (Fig. 4B).
A Gompit transformation linearized the ascospore production curves
from both controlled temperature and field ascospore collection data
(Fig. 5, 6A and 6B). The final two percent of the cumulative ascospore
production curves was not included in the regression equations. No
significant difference (P = .05) was found in the slopes of the
regression lines (k values) from controlled temperature and 1984 field
data Gompit transformations. These slopes were significantly different
from the slope of the regression lines from 1985 (Fig. 5, 6A and 6B).
A regression equation was developed from historic temperature data
to predict degree day accumulation from days elapsed from JD 90 (Fig.
7A). The equation closely predicted degree day accumulation in 1984 and
1985 (Fig. 7B) and can be used to predict degree day accumulation from the time ascospores first mature. Predicted degree day accumulation
allows prediction of cumulative spore production using the equation
developed from controlled chamber data (Fig. 5). Predicted ascospore
productivity using this technique is compared against actual ascospore
production for 1984 and 1985 (Fig. 8A and 8B).
Ascospore release
Ascospores were only collected in weeks with measurable
precipitation (Fig. 1 and 2). Hourly Burkhard spore trap data monitored 00
1984 1985 80 80
60
VASEUNE SPORE TRAP VASEUNE SPORE TRAP 40 ASCOSPORE TUNNEL ASCOSPORETUNNEL
20
0 400 800 1200 1600 0 400 800 1200 1600 2000 2400 DEGREE DAYS BASE 0 C DEGREE DAYS BASE 0 C
Figures 4A and 4B. Cumulative ascospore productivity from ascospore liberation tunnel and vaseline spore trap data plotted against degree days base 0 C for 1984 and 1985. Note degree day scales differ for the two years
I 38
5
4
m
Y=-2.39+.0066X • TRANSFORMED DATA
-3 0 200 400 600 800 1000 DEGREE DAYS BASE G C
Figure 5. Cumulative ascospore productivity from 9, 16, and 21 C controlled temperature cabinets transformed using a Gompit transformation of -ln(-ln(y)). R-square =0.96 for the regression line 5 1 1 1 1 1 5 T T ^ Y=-2.23+.0061X Y=-1.65+.0032X 4 OVASEUNE SPORE TRAP 9 o - 4 o VASEUNE SPORE TRAP • ASCOSPORE TUNNEL o / • ASCOSPORE TUNNEL p 3 — /o 3 • y6 1984 2 2 Uy4 1 1 8 / O 0 u/ 0 u / O LJ -1 -1 VO
-2 -2
-3 • 1 1 I • 1 1 •• I 1 I I ' 1 ' • • 1 ' • • -3 200 400 600 800 1000 1200 400 800 1200 1600 2000 DEGREE DAYS BASE 0 C DEGREE DAYS BASE 0 C
Figures 6A and 6B. Cumulative ascospore productivity data from 1984 and 1985 field seasons transformed using a Gompit transformation. R-square =0.96 for the 1984 regression and R-square =0.93 for the 1985 regression. Note degree day scales differ for the two years
I 2100 2000 "TO-
1800 Y=4.01+5.07X+0.14X-0.00036X^ o 1600 A 1500 i O ^ 1200 1200 I o ^ Oè° 900 800 o D APREDICTED a 01985 O • 1984 600 0^0 ! 400 300 o o •
' ,v.,I • ' ' I 111 • 11111 • I I • • • I • ' ' I • • • ' 30 60 90 120 100 120 140 160 180 200 220 DAYS ELAPSED JUUAN DATE -
Figure 7A. Predicted degree day accumulation from Julian date 90 based on historic temperature data. R-square =0.99
Figure 7B. Degree day accumulation predicted by the equation in 7A and observed degree day accumulation from the date of first ascospore collection in 1984 and 1985 1 r
100 - 0 O - 100 - 51519 0 o o
80 - 80 1985 / • o O
g 60 -I 60 g 40 - 40
g — PREDICTED 3 20 20 O VASEUNE SPORE TRAP O -PREDICTED • ASCOSPORE TUNNEL 0 - •0 o VASEUNE SPORE TRAP 0 • ASCOSPORE TUNNEL
_l L. -J 1 1 1 L 1 1 1 I I I I I I I I 100 120 140 160 180 100 120 140 160 180 200 220 JUUAN DATE JUUAN DATE
Figures 8A and 8B. Predicted ascoapore productivity using historic degree day data and the equation developed from controlled temperature cabinets (Fig. 5) plotted with actual ascospore productivity from 1984 and 1985 field seasons
I 42
in 1985 showed most ascospores were released after rains. Dew induced
release of low levels of spores on a few occasions during the peak spore
production period. Most wetting periods from rain and dew occurred
during the evening and early morning, although ascospore release was
usually delayed until daylight hours (Fig. 9). The majority of
ascospores were present in the air after 0600 hours and ascospore levels
peaked at 1000 hours (Fig. 9). In a laboratory experiment 19 percent of
the total spore release occurred 15 minutes after leaf discs were wetted
and release continued for 6 hours until leaf discs dried (Fig. 10).
Foliar and stem infection
Foliar infection of clone NC5272 occurred whenever measurable levels of ascospores were present during the period of 0-98% of total ascospore production in 1984 and 1985 (Fig. 11). Peak foliar infection periods corresponded to periods of peak ascospore collection (Fig. 1 and
2). Highest foliar infection ratings for the study also occurred within this period.
Stem infection of both clones exposed in the field in 1984 and 1985 were related to peak ascospore collection periods (Tables 3» 4, Fig. 1 and 2), before significant levels of conidia were trapped. Clone NC5271
(more resistant) was infected only during the weeks of peak ascospore production and was not infected during any other exposure periods in the field study (Tables 3 and 4). Mixed populations of ascospores and conidia were present after JD 146 in 1984 and JD 138 in 1985. Figure 9. Total number of ascospores collected by the Burkhard spore trap between Julian date 128 and 191 in 1985 and the total hours of wetness resulting from dew or rain in the same period E3 RAIN [33 OEM 15 1 r
10
5
0 7000
6000
5000
4000
3000
2000
1000
0 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ t^fiyaP?yaft^,TpifTprrp 600 1200 1800 HOUR OF THE DAY 45
100 -
80
60 -
40
20
0 2 3 4 5 6 HOURS AFTER WETTING
Figure 10. Percentage of total ascospores collected with the ascospore liberation tunnel after a single wetting. Leaf discs were completely dry between five and six hours after wetting o 1984 4 - • 1985 ci) z 3 -
Œ OC 2 - OC Œ
O 1 - Ov le
0 -
' ' ' ' ' I 1,1 I I I I » 100 120 140 160 180 200 JULIAN DATE
Figure 11. Foliar infection ratings of clone NC5272 during the period of 0-98% of total ascospore production in 1984 and 1985 field seasons. Foliar infection ratings were based on a scale of 0-5 where 0 = no Infection and 5 = 75+ lesions per leaf 47
Table 3. Stem infection of two Populus clones exposed in the field for 7 day periods during 0-98% of total ascospore production for 1984
Period ending Clone NC5272 Clone NC5271 on Julian Date No. cankers/stem No. cankers/stem i% infected) (Î infected)
118 0.00(0) 0.00(0)
125 0.00(0) 0.00(0)
132 0.00(0) 0.00(0)
139 0.00(0) 0.00(0)
146 2.30(93) 0.22(22)
153 0.00(0) 0.00(0)
160 0.00(0) 0.00(0)
174 0.33(33) 0.00(0)
181 0.27(27) 0.00(0) 48
Table 4. Stem infection of two Populus clones exposed in the field for 7 day periods during 0-98% of total ascospore production for 1985
Period ending Clone NC5272 Clone NC5271 on Julian Date No. cankers/stem No. cankers/stem (% infected) (% infected)
114 0.00(0) 0.00(0)
121 0.00(0) 0.00(0)
128 0.40(40) 0.11(11)
135 1.30(60) 0.22(22)
142 0.06(6) 0.00(0)
149 0.00(0) 0.00(0)
156 0.20(20) 0.00(0)
163 0.06(6) 0.00(0)
170 0.66(46) 0.00(0)
177 0.00(0) 0.00(0)
184 0.00(0) 0.00(0)
191 0.00(0) 0.00(0) 49
Effect of the environment on spore germination and infection
Ascospores germinated over a wide range of temperatures although no
germination occurred at 35 C and was negligible at 5 C after 16 hours
incubation (Fig. 12A and 12B). Germination was greater than 40 percent
after 4 hours at 20, 25, and 30 C (Fig. 12B). Most spores germinated by
polar germ tubes and the number of germ tubes averaged 1.0, 1.0, 1.08,
1.36, and 1.5 after 16 hours at 10, 15, 20, 25 and 30 C, respectively.
Correlation coefficients between weekly foliar and stem infection levels and environmental and spore data were determined (Tables 5-7).
Ascospore densities were highly correlated with foliar and stem infection data, except for stem infection in 1984 (Tables 5-7).
Correlations with the environment showed that hours of wetness when wetting periods were initiated between 0700 and 1900 were generally better correlated with foliar and stem infection than were longest wetting period or total hours of wetness in a week. Interactions between hours of wetness and average temperature during the wetting period were also highly correlated with levels of the disease while temperature effects alone were not (Tables 5-7).
Ascospore densities measured by the vaseline spore trap and measures of total hours wetness initiated between 0700 and 1900 were most useful in developing regression equations. The interactions between total hours of wetness and average temperature during the total hours wetting or combinations of total hours wetness and average temperature during the total hours wetness generally provided better regression equations than if total hours of wetness or average temperature during wetting were used alone (Tables 8-10). A ipÇ O 15^ 100 - A 10 c O 20_C o 15 c • 25_C O 20 C 30 C 2100 80 - • 25 C
60 -
- on 40 -
20
8 12 8 12 16 HOURS HOURS
Figures 12A and 12B. Germ tube elongation and percentage germination of ascospores discharges from overwintered leaves onto water agar. Each data point Is the average of five replications of 125 spores per replication Table 5. Correlation coefficients between weekly foliar and stem infection data and spore trap and environmental parameters measured during the period of 0-98% of the total ascospore productivity in the field for 1984
Foliar Infections % stem Infections Vaseline rating per stem infected per stem spore trap (NC5272) (NC5272) (NC5271)
Vaseline spore trap .685* .557 .438 .610 - Longest wet® .418 .362 .554 .232 -.037 Total wet® .312 .356 .558 .437 -.121 Total wet 0700-1900 .760** .894*** .924*** .847** .366 Temp longest wet° .274 .356 .505 .251 .056 Temp total wet° . .304 .497 .534 .273 .076 Temp X longest wet .606 .409 .629 .260 -.055 Temp X total wet- .689* .602 .744 .498 .083 Temp X total wet 0700-1900 .784** .718* .856** .611 .211
^Longest and total hours of wetness in a 7-day sampling period. Total hours wetness when wetting occurred between 0700-1900 hours. ^Average temperature during longest and total wetting periods. Interaction between longest period of wetness and average temperature during the wetting period. Interaction between total hours of wetness and average temperature during these wetting periods. Interaction between number of hours wetness when wetting periods occurred between 0700- 1900 hours and average temperature during the wetting periods. ^Significant at P = .05. **Signiflcant at P = .01. ***Significant at P = .001. Table 6. Correlation coefficients between weekly foliar and stem infection data and spore trap and environmental parameters measured during the period of 0-98% of the total ascospore productivity in the field for 1985
Foliar Infections % stem Infections Vaseline rating per stem infected per stem spore trap (NC5272) (NC5272) (NC5271)
Vaseline spore trap .722** .940*** .944*** .455 - Longest wet .729** .245 .264 .199 .396 Total wet^ .848*** .585* .607* .323 .721** Total wet 0700-1900 .797*** .419 .453 .102 .537 Temp longest wet° .200 .014 .001 -.123 -.001 Temp total wet° . .467 .206 .245 .033 .258 Temp X longest wet .691 A* .182 .181 .053 .272 Temp X total wet® .920*** .651* .682** .284 .759** Temp X total wet 0700-1900 .881*** .512 .551* .021 .584*
.Longest and total hours of wetness in a 7-day sampling period. Total hours wetness when wetting occurred between 0700-1900 hours. ^Average temperature during longest and total wetting periods. Interaction between longest period of wetness and average temperature during the wetting period. Interaction between total hours of wetness and average temperature during these wetting periods. Interaction between number of hours wetness when wetting periods occurred between 0700- 1900 hours and average temperature during the wetting periods. *Significant at P = .05. **Significant at P = .01. ***Significant at P = .001. Table 7. Correlation coefficients between weekly foliar and stem infection periods and spore density and selected environmental parameters measured in the field for the period of 0-9855 of total ascospore productivity for 1984 and 1985 combined
Foliar Infections % stem Infections Vaseline rating per stem infected per stem spore trap (NC5272) (NC5272) (NC5271)
Vaseline spore trap 700*** .687*** .661*** .523*** — Longest wet 622** .321 .410 .187 .198 Total wet® . 522** .467* .486* .292 .215 Total wet initiated 0700-1900 789*** .720*** .713*** .462* .447* Temp longest wet° 488* .279 .382 .141 .147 Temp total wet° , 339 .184 .249 .052 -.001 Temp X longest wet 648*** .353 .471* .161 .091 Temp X total wet® 748*** .595** .671*** .357 .300 Temp X total wet 0700-1900 808*** .666*** ,742*** .360 .339
^Longest and total hours of wetness in a 7-day sampling period. Total hours wetness when wetting occurred between 0700-I900 hours. ^Average temperatures during total and longest wetting periods. Interaction between longest period of wetness and average temperature during the wetting period. Interaction between total hours of wetness and average temperature during these wetting periods. Interaction between number of hours wetness when wetting periods occurred between 0700- 1900 hours and average temperature during the wetting periods. ^Significant at P = .05. **Significant at P = .01. ***Signifioant at P = .001. Table 8. Regression models developed to relate foliar and stem infection levels during 0-98% of asoospore productivity to ascospore densities and the environment monitored in 1984. All variables used in the regression models are significant at P = .05
Dependent Intercept Variablel Variables Variables F variable (RO® (RC) (RC)
Foliar 0.17 Ascospore density^ Total hours® Temp total^ 92.7* 0.98 rating wet 0700-1900 wet (0.008) (0.032) (0.092)
Infections -0,68 Total hours wet° 28.2* 0.80 per stem 0700-1900 NC5272 (0.047)
^Regression coefficient,
'^Ascospore density monitored by vaseline spore traps.
^Total hours of wetness when wetting periods occurred between 0700-1900 hours.
^Average temperature during total hours wetness for a T-day period.
^Significant at P = .001. Table 9. Regression models developed to relate foliar and stem infection levels during 0-98% of ascospore productivity to asoospore densities and the environment monitored in 1985. All variables used in the regression models are significant at P = .05
2 Dependent Intercept Variablel Variables F R variable (RC)^ (RC)
Foliar 0,03 Ascospore density^ Temp X total wet° 24.06* 0.84 rating 0700-1900 (0.004) (0.004)
Infections -0.12 Ascospore density - 76.5* 0.88 per stem (0.007) NC5272
^Regression coefficient.
^Ascospore density monitored by vaseline spore traps.
^Interaction between total number of hours wetness when wetting periods occurred between 0700-1900 hours and average temperature during the wetting period.
'•«Significant at P = .0001. Table 10. Regression models developed to relate foliar and stem infection levels during 0-98% of ascospore productivity to ascospore densities and the environment for 1984 and 1985 combined. All variables used in the regression models are significant at P = .05
Dependent Intercept Variablel Variable2 variable (RC)8 (RC)
Foliar 0.14 Ascospore density Temp X total wet 54.8* 0.86 rating 0700-1900 (0.008) (0.002)
Infections -0.38 Ascospore density Total wet^ 19.6* 0.69 per stem 0700-1900 NC5272 (0.005) (0.0211)
^Regression coefficient.
^Ascospore density monitored by vaseline spore traps.
^Interaction between total number of hours wetness when wetting periods occurred between O70O-I90O hours and average temperature during the wetting period.
^Total hours wetness when wetting periods occurred between 0700-1900 hours.
^Significant at P = .001. 57
DISCUSSION
Ascospore productivity by Mycosphaerella populorum exhibited a
distinct seasonal periodicity that commenced in April, peaked in mid-to
late May and declined to low levels until no spores were collected in
early August in 1984 and September in 1985. Initial seasonal ascospore
production coincided approximately with the time of bud swell on Populus
deltoides in central Iowa (Table 2). Selection of a phenophase, such as
bud swell on deltoides, allowed a reliable assessment of ascospore
development in the field when laboratory determinations could not be
made (MacHardy and Gadoury, 1985). Identification of a phenophase
between Drepanopeziza punctiformis and Populus species in the
Netherlands and Venturia inaequalis and apple in the northeastern United
States facilitated initiation of fungicide sprays to cover primary
infection periods (de Kam, 1975; MacHardy and Gadoury, 1985; Szkolnik,
1969). The apple scab phenophase, silver tip on the variety Mcintosh,
also served as a biofix for the initiation of a model that predicted
ascospore population development (MacHardy and Gadoury, 1985). Temporal
differences between ascospore development and the selected phenophase
were not uncommon in the inaequalis-McIntosh system, although in most
years 1% of the ascospores were mature by the silver tip developmental
stage (MacHardy and Gadoury, 1985). Temporal differences between bud swell and ascospore collection were evident in this study. Yearly
evaluation over an extended period would be required to firmly establish the reliability of the cottonwood bud swell phenophase for estimating 58
ascospore development of populorum.
Ascospore productivity of ^ populorum was related to degree day
accumulation (base 0 C) after ascospores were first collected. Three
phases of ascospore productivity could be identified in the cumulative
ascospore productivity-degree day curves. The first phase was a short
lag period (0-5% of total productivity), the second was a phase of rapid
or accelerated productivity (6-98% of total productivity) and the third or final phase was an extended period of low productivity (99-100% of total productivity) that encompassed a large portion of the growing season (Fig. 3B, 4A and 4B). Three distinct phases were also identified in cumulative ascospore maturation-degree day curves of inaequalis
(MacHardy and Gadoury, 1985).
The curves developed for the scab fungus exhibited a lag (0-10% of total maturation) and final phase (91-100% of total maturation) of nearly equal duration and were symmetric about their inflection point.
The short lag phase and the extended period of ascospore productivity by
M. populorum resulted in ascospore productivity-degree day curves that were asymmetric about their inflection point and skewed to the right.
Similar asymmetric growth curves in other biologic and pathosystems were best described by a Gompertz equation (Berger, 1981; Chinchilla-Lopez,
1985). The Gompertz transformation from this equation (Berger, 1981) linearized the ascospore productivity-degree day curves in this study.
The final two percent of total cumulative ascospore productivity was not included in the Gompit transformations.
Ascospores were produced over a period that spanned 5-6 months 59
(Fig. 1 and 2; Thompson, 1941). Factors other than degree days probably
affected the final phase of ascospore productivity (99-100%) which
extended for a 1-2 month period (Fig. 1, 2 and 3A). Dry spring weather
in 1985 (Fig. 2) and in a study by Thompson (1941) were related to an
extended ascospore production period. Gerstenberger (1984), however,
found that ascospore production period was shorter in a dry year in
comparison to a wet year. Moisture availability (James and Sutton,
1982b) and rainfall frequency (Hirst and Stedman, 1961) affected the
duration of ascospore production of inaequalis. These factors and
others such as leaf tissue degradation which differed greatly in the two
field seasons (Fig. 22) may have affected the duration of ascospore
production by ^ populorum. Utilization of degree days alone to
estimate the extended period of ascospore productivity would therefore
have resulted in significant deviations from predicted values.
Cumulative ascospore productivity-degree day curves from controlled temperature chambers and 1984 field data were similar. Significant differences from these data were found in 1985 productivity curves. Dry spring weather (driest in 112 years) probably decreased the ascospore productivity rate in 1985. A lack of moisture delayed ascospore maturation of inaequalis in certain years in North Carolina (James and Sutton, 1982b) and New Hampshire (MacHardy and Gadoury, 1985). Dry weather potentially affected both the rate and duration of ascospore production by ^ populorum in 1985.
Differences in the rate and period of peak ascospore production were evident between the ascospore liberation tunnel and the vaseline 60
spore trap. Peak spore production periods were later (Fig. 1 and 2) and the rate of ascospore production slower (Fig. 4A and 4B) when monitored by the vaseline spore trap. Differences between the spore trap methods may reflect a difference in the frequency and duration Of ascospore release under field and laboratory conditions. Ascospore release was induced on a regular basis by wetting samples and ascospores were collected for a set time period each week in ascospore liberation tunnel experiments. Ascospore release under field conditions was dependant on the frequency, timing and duration of wetting periods resulting from rain (Fig. 1, 2 and 9). Ascospores produced under field conditions would not be collected until released by wetting. This difference between time of ascospore production and release may have been reflected in the slower rate and later period of peak ascospore productivity monitored by the vaseline spore trap.
Utilization of historic degree day data and cumulative ascospore productivity-degree day curves developed from controlled temperature incubation allowed prediction of ascospore productivity after ascospores first matured. The same approach predicted ascospore maturation of V. inaequalis during six growing seasons in New Hampshire (MacHardy and
Gadoury, 1985). Deviations from predicted ascospore maturation of V. inaequalis (MacHardy and Gadoury, 1985) and ascospore productivity of M. populorum in this study occurred in years with abnormally dry weather during the accelerated phase of ascospore development. Continued monitoring of cumulative ascospore productivity-degree day relationships in multiple years and locations will allow better assessment of 61
prediction of ascospore development using degree days.
Release of ascospores from pseudothecia of ^ populorum was
dependant upon wettings from rain. Thompson (1941) and Gerstenberger
(1983) also collected the majority of ascospores after periods of rain.
Dew periods were common during the period of 0-98% of total ascospore productivity (Fig. 9)« However, relatively few ascospores were released by dew in comparison to rain. A relationship between hour of the day and ascospore release also was evident from hourly Burkhard spore trap collections (Fig. 9). Most ascospores of M. populorum were trapped during daylight hours, although wettings from dew and rain did not show a similar distribution (Fig. 9). A similar relationship was present between ascospore release and hour of the day for inaequalis (Hirst and Stedman, 1962). Brook (1969a) found far red light stimulated ascospore release while infrared light suppressed release by V. inaequalis. Suppression of ascospore release under light and dark conditions was evident in this study. Laboratory experiments demonstrated that under daylight conditions large numbers of ascospores were released a short time after wetting (Fig. 10). However, in periods when wetting from rain occurred at night in the field, ascospores were not released until daylight hours (Fig. 9). Further study of M. populorum and other Mycosphaerella species that exhibit a diurnal periodicity of ascospore release (Carter, 1963; Meredith et al., 1973;
Whiteside, 1970; Meredith, 1962) may show a specific effect of light on ascospore discharge.
Foliar infection of clone NC5272 occurred whenever measurable 62
levels of ascospores were detected with the vaseline spore trap. These
data support observations by Waterman (1954) and Bier (1939) that
primary infection of Populus foliage in the spring is due to the flux of
ascospores released from pseudothecia in overwintered leaves. Infection ratings were also highly correlated with ascospore densities. Peak foliar infection levels corresponded to peak ascospore collection periods in the field. A majority of primary infection therefore occurred during the accelerated phase of ascospore production when peak ascospore inoculum levels existed.
Correlation and regression analysis showed that environmental parameters measured during the period of 0-98% of total ascospore production also modified foliar infection ratings (Tables 5-10).
Interaction between hours of wetness and average temperature during wetting periods (longest, total and those during 0700-1900 hours) were generally the best correlated with foliar infection ratings. Levels of foliar infection at specific temperature-time combinations were not determined. However, ascospores of populorum germinated slowly at 10
C (Fig. 12A and 12B) and below. Ascospore penetration of stomata (Niyo et al., 1986) during wetting periods at 10 C or below may require extended periods of free moisture to cause significant levels of infection. A relationship between foliar infection and average temperature and duration of wetting was used to predict the severity of primary infection by V. inaequalis (Mills, 1944). Similar data would be useful in prediction of foliar infection severity caused by populorum under various temperature-wetting period duration combinations. 63
Wetting periods during daylight hours and the interaction with
average temperature during these wetting periods were highly correlated
with foliar infection ratings (Tables 5-7). Ascospore release occurred
primarily during daylight hours (Fig. 9). Wetting periods from rain
during daylight hours probably resulted in higher foliar infection
levels due to higher ascospore densities. Wetting periods from rain
during the night presumably resulted in lower infection levels since
suppression of ascospore release occurred at these times.
Stem infection of clones NC5271 and NC5272 was found over a
narrower range of inoculum levels than was foliar infection. Stem
infection periods were restricted mainly to periods of peak ascospore
collection monitored by the vaseline spore trap. Highest levels of stem
infection of NC5272 and the sole infection periods of clone NC5271
occurred during the period of peak ascospore production. These
infection periods could be attributed to ascospores since significant
levels of conidia were not trapped. Filer et al. (1971) found 60% of
the stems of a susceptible P_^ deltoides were infected after exposure to
overwintered leaves with ^ populorum fruiting bodies. McNabb et al.
(1982) suggested that multiple infection of stems in a dense stand may
have resulted from the accumulation of leaf litter and subsequent high
ascospore levels around stems. Ascospore densities monitored by the
vaseline spore trap in this study were highly correlated with stem infection levels (Table 5-7). Primary infection of Populus stems in the spring in this study were related to peak ascospore release periods that occurred during the accelerated phase of ascospore production. 64
Environmental affects on ascospore infection of stems were similar to that found for foliar infection (Table 5-10). Environmental parameters, however, were usually less correlated with foliar and stem infection levels than were correlations with foliar disease ratings.
Wetting periods during daylight hours and the interaction between wetting period duration and average temperature were the best correlated and contributed most frequently to regression models. Wetting periods that occurred during daylight hours and the period of peak ascospore release are probably the most important in the build up of stem disease.
Results from this study indicated that these conditions occurred on a periodic basis during the accelerated phase of ascospore production. 65
LITERATURE CITED
Arnold, C. Y. I960. Maximum-minimum temperatures as a basis for computing heat units. Proc. Am. Soc. Hortic. Soi. 76:682-692.
Barr, M. E. 1972. Preliminary studies on the Dothideales in temperate North America. Contrib. from the Univ. Mich. Herb. 9:523-638.
Berger, R. D. 1981. Comparison of the Gompertz and logistic equations to describe plant disease progress. Phytopathology 71:716-719.
Berger, R. D. 1977. Application of epidemiological principles to achieve plant disease control. Ann. Rev. Phytopathol. 15:165-183.
Bier, J. E. 1939. Septoria canker of introduced and native poplars. Can. J. Res. 7:195-204.
Brook, P. J. 1969a. Stimulation of ascospore release in Venturia inaequalis by far red light. Nature 222:390-392.
Brook, P. J. 1969b. Effects of light, temperature, and moisture on release of ascospores by Venturia inaequalis. N. Z. J. Agric. Res. 12:214-217.
Burchill, R. T. and K. E. Hutton. 1968. The suppression of ascospore production to facilitate the control of apple scab (Venturia inaequalis (Cke.) Wint.). Ann. Appl. Biol. 56:285-292.
Carlson, L. W. 1974. Fungicidal control of poplar leaf spots. Can. Plant Dis. Surv. 54:81-65.
Carlson, L. W. 1972. Fungicidal control of poplar leaf spots in Alberta and Saskatchewan. Can. Plant Dis. Surv. 52:99-101.
Carter, M. V. 1963. Mycosphaerella pinoides. II. The phenology of ascospore release. Aust. J. Biol. Sci. 16:800-817.
Chinchilla-Lopez, C. 1985. Pathogen survival and host resistance in eyespot disease of maize caused by Kabatiella zeae Narita and Hiratuska. Ph.D. Dissertation, Iowa State University, Ames Iowa.
Cook, R. T. A. 1974. Pustules on wood as sources of inoculum in apple scab and their response to chemical treatments. Ann. Appl. Biol. 77:1-9.
Cooper, D. T. and T. H. Filer. 1976. Resistance to Septoria leaf spot in eastern cottonwood. Plant Dis. Rept. 60:812-814. 65
de Kam, M. 1975. Ascospore discharge in Drepanopeziza punctiformis in relation to infection of some poplar clones. Eur. J. For. Pathol. 5:304-309.
Dennis, R. W. G. 1968. British ascomycetes. Second ed. J. Cramer, Lehre, Germany.
Ellis, J. B. and B. M. Everhart. 1892. The North American pyrenomycetes. Ellis and Everhart, Newfield, New Jersey.
Filer, T. H., F. I. McCraken, C. A. Mohn and W. K. Randall. 1971. Septoria canker on nursery stock of Populus deltoïdes. Plant Dis. Rept. 55:460-463.
Ford, H. F. and A. M. Waterman. 1954. Effect of surface sterilization on survival and growth of field-planted hybrid poplar cuttings. Plant Dis. Rep. 38:101-105.
Gadoury, D. M. and W. E. MacHardy. 1986. Forecasting ascospore dose of Venturia inaequalis in commercial apple orchards. Phytopathology 76:112-118.
Gadoury, D. M. and W. E. MacHardy. 1982a. Effects of temperature on the development of pseudothecia of Venturia inaequalis. Plant Dis. 66:464-468.
Gadoury, D. M. and W. E. MacHardy. 1982b. A model to estimate the maturity of ascospores of Venturia inaequalis. Phytopathology 72:901-904.
Gerstenberger, P. E. 1983. Epidemiology of Septoria musiva (Mycosphaerella populorum) in intensive culture plantations of poplar. Unpublished M.S. Thesis. Iowa State University.
Gillespie, T. J. and G. E. Kidd. 1973. Sensing duration of leaf moisture using electrical resistance. Can. J. PI. Sci. 58:179-187.
Gilpatrik, J. D. 1978. Single application treatments for apple scab control. Proc. Apple and Pear Scab Workshop. N. Y. Geneva Agric. Exp. Stn. Spec. Rep. 28:30-32
Gilpatrick, J. D. and M. Szkolnik. 1978. The maturation and discharge of ascospores of the apple scab fungus. Proc. Apple and Pear Scab Workshop. N. Y. Geneva Agric. Exp, Stn. Spec. Rep. 28:1-6.
Hare, W. W. and J. C. Walker. 1944. Ascochyta disease of canning peas. Wis. Agric. Exp. Stn. Res. Bull. No. 150.
Higgins, B. B. 1936. Morphology and life history of some ascomycetes 67
with special reference to the presence and function of spermatia. III. Am. J. Bot. 23:598-602.
Higgins, B. B. 1929. Morphology and life history of some ascomycetes with special reference to the presence and function of spermatia. II. Am. J. Bot. 16:287-297.
Hirst, J. M. 1953. Changes in atmospheric spore content: Diunrnal periodicity and the effects of weather. Trans. Br. Mycol. Soc. 36:375-393.
Hirst, J. M. 1952. An automatic volumetric spore trap. Ann. Appl. Biol. 39:257-265.
Hirst, J. M and 0. J. Stedman. 1962. The epidemiology of apple scab Venturia inaequalis (Cke.) Wint.). II. Observations on the liberation of ascospores. Ann. Appl. Biol. 50:525-550.
Hirst, J. M. and 0. J. Stedman. 1961. The epidemiology of apple scab (Venturia inaequalis (Cke.) Wint.). I. Frequency of airborne spores in orchards. Ann. Appl. Biol. 49:290-305.
Ingold, C. T. 1961. The biology of fungi. Hutchinson Educational, London.
James, J. R. and T. B. Sutton. 1982a. Environmental factors influencing pseudothecial development and ascospore maturation of Venturia inaequalis. Phytopathology 72:1073-1080.
James, J. R. and T. B. Sutton. 1982b. A model for predicting ascospore maturation of Venturia inaequalis. Phytopathology 72:1081-1085.
Jenkins, W. A. 1938. Two fungi causing leaf spots of peanut. J. Agric. Res. 56:317-332.
Jones, A. L. 1978. Analysis of apple scab epidemics and attempts at improved disease prediction. Proc. Apple and Pear Scab Workshop. N. Y. Geneva Agric. Exp. Stn. Spec. Rep. No. 28:19-22.
Lewis, F. H. 1980. Control of deciduous tree fruit diseases: A success story. Plant. Dis. 64:258-263.
Luley, C. J. and P. D. Manion. 1984. Inoculum potential of Gremmeniella abietina in New York. Pages 82-95. In P. D. Manion, ed. Scleroderris canker of conifers. Nijhoff-Junk, Boston.
Luttrell, E. S. 1973. Loculoascomycetes. Pages 135-219. In G. C. 68
Ainsworth, K. F. Sparrow and A. S. Sussman, eds. The Fungi Vol. IVA. Academic Press, New York.
MacHardy, W. E. and D. M. Gadoury. 1985. Forecasting the seasonal maturation of ascospores of Venturia inaequalis. Phytopathology 75:381-385.
Manion, P. D. 1981. Tree disease concepts. Prentice Hall, Englewood Cliffs, New Jersey,
Massie, L. B. and M. Szkolnik. 1974. Prediction of ascospore maturation of Venturia inaequalis utilizing cumulative degree days. Abstr. Proc. Am. Phyt. Soc. 1:140.
Meredith, D. S. 1962. Some components of the air spora in Jamaican banana plantations. Ann. Appl. Biol. 50:577-59%.
Meredith, D. S., J. S. Lawerence and I. D. Firmann. 1973. Ascospore release and dispersal in black leaf streak disease of bananas (Mycosphaerella fijiensis). Trans. Br. Mycol. Soc. 60:547-554.
McGee, D. C. 1977. Black leg (Leptosphaeria maculans (Desm.) Ces. et de Not.) of rapeseed in Victoria: Sources of infection and relationships between inoculum, environmental factors and disease severity. Aust. J. Agric. Res. 28:53-62.
McGee, D. C. and G. A. Petrie. 1979. Seasonal patterns of ascospore discharge by Leptosphaeria maculans in relation to blackleg of oilseed rape. Phytopathology 69:586-589.
McNabb, H. S., Jr., M. E. Ostry, R. S. Sonneliiter and P. E. Gerstenberger. 1982. The effect and possible integrated management of Septoria musiva in intensive, short rotation culture of Populus in the north central states. Pages 51-58. In J. Zavitokovski and E. A. Hansen, eds. Proc. N. Am. Poplar Counc. Meet. Kansas State University.
Mills, W. D. 1944. Efficient use of sulfer dusts and sprays during rain to control apple scab. Cornell Agric. Exp. Stn. Ext. Bull. 630.
Niyo, K. A., H. S. McNabb, Jr. and L. H. Tiffany. 1986. Ultrastructure of the ascocarps, asci and ascospores of Mycosphaerella populorum. Myologia 78:202-212.
Nyvall, R. F. 1979. Field crop diseases handbook. AVI Publishing Co., Westport, Conn.
O'Leary, A. L. and T. B. Sutton. 1986. The influence of temperature and moisture on the quantitative production of pseudothecia of 69
Venturia inaequalis» Phytopathology 76:199-204.
Ostry, M. E. and H. S. McNabb, Jr. 1985. Susceptibility of Populus species and hybrids to disease in the north central states. Plant Dis. 69:755-757.
Ostry, M. E. and H. S. McNabb, Jr. 1982. Preventing blackstem damage to Populus hardwood cuttings. Pages 36-43. In J. Zavitkowski and E. A. Hansen, eds. Proc. N. Am. Poplar Counc Ann. Meet., Kansas State University.
Ostry, M. E. and T. H. Nicholls. 1982. A technique for trapping fungal spores. USDA For. Serv. N. C. For. Res. Note NC-283.
Palmer, M. E., A. L. Schipper, Jr. and M. E. Ostry. 1980. How to identify Septoria leaf spot and canker of poplar. USDA N. C. For. Exp. Stn., St. Paul, Minn.
Peck, C. H. 1884. Ann. Rep. N. Y. St. Bot. 35:138.
Ross, R. G. and S. A. Hamlin. 1962. Production of perithecia of Venturia inaequalis on sterile apple leaf discs. Can. J. Bot. 40:629-635.
Ross, R. G and R. J. Newberry. 1975. Effects of seasonal fungicide sprays on perithecium formation and ascospore production in Venturia inaequalis. Can. J. Plant Sci. 55:737-742.
Saccardo, P. A. 1915. Notae mycologicae. Ann. Mycol. 13:115-146.
Sanderson, F. R. 1977. Mycosphaerella graminicola (Fuckel) Sanderson comb, nov., the ascogenous state of Septoria tritici Rob. and Desm. New Z. J. Bot. 14:359-360.
Sanderson, F. R. and J. G. Hampton. 1978. Role of the perfect states in the epidemiology of the common Septoria diseases of wheat. New Z. J. Agric. Res. 21:277-281.
Sanderson, F. R., A. L. Scharen and P. R. Scott. 1985. Sources and importance of primary infection and identities of associated propagules. USDA Agric. Res. Serv. ARS-12:57-64.
Sarasola, A. A. 1944. Dos septoriosis de las alamedas Argentinas. Rev. Argen. Agron. 11:20-43. Abstr. In Rev. Appl. Mycol 23:365-366. 1944.
Seem, R. C. and S. P. Eisensmith. 1986. What's wrong with the Julian day. Phytopathology 76:41. 70
Sutton, T. B. 1978. Role of conidia of Venturia inaequalis in the epidemiology of apple scab. In Proc. Apple and Pear Scab Workshop. N. Y. Geneva Agric. Exp. Stn. Spec. Rep 28:6-9.
Sutton, T. B., J. R. James and J. F. Nardacci. 1981. Evaluation of a New York ascospore maturity model for Venturia inaequalis in North Carolina. Phytopathology 71:1030-1032.
Szkolnik, M. 1969. Maturation and discharge of ascospores of Venturia inaequalis. Plant Dis Rep. 53:534-537.
Thompson, G. E. 1941. Leaf spot diseases of poplars caused by Septoria musiva and ^ populicola. Phytopathology 31:241-254.
Van Der Plank, J. E. 1963. Plant disease: epidemics and controls. Academic Press, New York.
Waterman, A. M. 1954. Septoria canker of poplars in the United States. USDA Cir. 947.
Waterman, A. M. and K. F. Aldrich. 1952. Surface sterilization of poplar cuttings. Plant Dis. Rep. 36:203-207.
Whiteside, J. 0. 1970. Etiology and epidemiology of citrus greasy spot. Phytopathology 60:1409-1414.
Wilson, E. E. 1928. Studies of the acigerous stage of Venturia inaequalis (Cke.) Wint. in the relation to certain factors of the environment. Phytopathology 18:375-418.
Zadoks, J. C. and R. D. Schein. 1979. Epidemiology and plant disease management. Oxford University Press, New York.
Zalasky, H, 1978. Stem and leaf infections caused by Septoria musiva and ^ populicola on poplar seedlings. Phytoprotection 59:43-50. 71
SECTION II. CONIDIAL RELEASE, GERMINATION AND INFECTION OF POPDLUS BY SEPTORIA MDSIVA 72
INTRODUCTION
Intensification of most foliar diseases during the growing season
is due to repeating generations of an asexual state. Each asexual
generation is characterized by a series of concatenated processes that
make up the secondary disease cycle. Frequently, this chain of events
is quantified by single parameters such as disease increase rates or
changes in levels of pathogen propagules during the growing season. It
is also possible to quantify each event of the secondary disease cycle
separately, and this is the theoretical basis for mathematical modeling
of disease epidemics (Kranz, 1979).
Most studies of Septoria leaf spot and canker have evaluated
disease levels in response to various treatments under field conditions.
No studies have established treatment effects on specific events such as
spore release, spore germination, infection of host tissues, incubation
periods or other aspects of the secondary disease cycle. For this
reason, it has often been difficult to explain changes in disease
severity that have resulted from silvicultural treatments (Bowersox and
Merrill, 1976). Therefore, quantification of the various aspects of the
disease cycle and of factors that influence disease intensification
during the growing season were warranted. The research objectives of this section were to (1) quantify asexual spore release and infection of two Populus clones under field conditions (2) determine the effect of environmental factors on asexual spore release and infection (3) quantify the effect of temperature, time and moisture on spore 73
germination and on leaf and stem infection under controlled conditions
and (4) determine the effect of inoculum density on infection of stems and leaves. 74
LITERATURE REVIEW
Symptoms of Septoria leaf spot and canker are usually attributed to infection by the asexual state Septoria musiva Peck, which is commonly isolated from stems and leaves during the growing season. Although Peck
(1884) described S. musiva from the living leaves of Populus deltoides over 100 years ago, there have been few studies on the role of the conidia in foliar and stem disease build up. Since studies of the factors affecting conidial production, release, dispersal and infection of leaves and stems are limited, the following review includes information from other Septoria species and pathosystems.
The Fungus
Septoria musiva is a pycnidial fungus with cylindrical, straight or slightly curved, hyaline, septate conidia (Thompson, 1941; Palmer et al., 1980). One to four septa per conidium is apparently most common
(Thompson, 1941; Palmer et al., 1980) although six septa have been reported (Waterman, 1954; Davis, 1915). Conidial dimensions are modified by the host on which they are produced (Thompson, 1941;
Waterman, 1954). Thompson (1941) reported conidial measurements of 28-
54 X 3.5-4 pm. Palmer et al. (1980), Waterman (1954), and Bier (1939) indicated a length range of 17-56 ;um is inclusive. The wider range indicated by the latter measurements may be attributed to inclusion of atypical conidia (noted by Thompson (1941)) produced in pycnidia in small, white to gray leaf spots. Conidia from such lesions were 0 to 2- 75
septate, elliptical-fusiform or cylindrical and measured 12-29 x 3.5-
4ym. Colonies produced from these spores sporulate slowly and sparingly
(Thompson, 1941).
Vegetative growth occurs on most common media. Growth optimum on
potato dextrose agar was reported to be 27 C (Thompson, 1941), although
growth at other temperatures was not reported. Sporulation may vary
significantly among media, and spore production occurs during a short
time period in colony growth (Waterman, 1954). Single spore isolates
often yield colonies that produce only vegetative mycelium (Waterman,
1954) suggesting that changes in colony characteristics occur readily in
culture.
Inoculum Production
Pycnidia of ^ musiva are produced in both stem and leaf tissue
throughout the growing season (Bier, 1939; Waterman, 1954). In leaves,
pycnidia develop on both leaf surfaces (Thompson, 1941) and produce
conidia soon after symptoms appear in the spring (Bier, 1939), or
approximately early June in central Iowa (Young et al., 1980).
Ostioles of ^ musiva pycnidia either protrude from embedded pycnidia or
open widely giving the pycnidium the appearance of an acervulus in leaf tissue (Thompson, 1941; Waterman, 1954). Spores are exuded in pinkish
masses or long spore tendrils which turn white on drying (Thompson,
1941; Waterman, 1954).
Pycnidia in cankers may be buried in host tissue or produced within lenticels in thick, black stroma-like fungal tissues (Waterman, 1954). 76
Pycnidial production in cankers is ephemeral and is rarely observed on
stem lesions two or more years of age (Bier, 1939; Waterman, 1954). The
short period of pycnidial production is probably related to invasion of
cankers by secondary fungi. Secondary pathogens of various genera and
nonpathogenic fungi are often isolated from two year and older cankers.
These fungi eventually displace S. musiva in stem tissues (Filer, 1971)
and apparently also inhibit sporulation of ^ musiva.
Overwintering of Conidia
Conidia of the imperfect state that survive the winter are a
potential source of primary infection in the spring. The year following
infection. Bier (1939) collected branches with pycnidia containing
viable conidia. McNabb et al. (1982) indicated pycnidia in overwintered
leaves are another potential source of primary inoculum. The viability
of overwintered pycnidia of ^ musiva in leaves and stems and their
importance as a source of primary inoculum are not known. However,
conidia of Marssonina brunnea (E.and E.) Magn. from twig pustules are a
source of primary foliar infection of poplars in France (Pinon and
Poissonnier, 1975). Overwintered pycnidia and mycelium of S. glycines
Hemmi. (Anonymous, 1982), ^ tritici Rob. ex. Desm. (Sanderson and
Hampton, 1978), and S. lycopersici Speg. (Chuppe and Scherf, I960) are
the main sources of primary inoculum for these foliar pathogens. In
addition, conidia of inaequalis can overwinter in infected branch tissue and initiate primary leaf infections in the spring (Cook, 1974).
This source of overwintering inoculum can be important early in the 77
year, before ascospores mature. Fungicide sprays that are applied
solely on the basis of ascospore maturity may miss primary infections
caused by overwintered conidia (Sutton, 1978). A similar role of
conidia of S. musiva in initiating early spring foliar infection is
possible.
Conidia Release and Dispersal
Gerstenberger (1983) found that release of conidia occurred only
after rain. As little as 0.13 cm of rain was found to be sufficient to
induce spore release. Wet growing seasons are apparently conducive to
production and release of large numbers of conidia because larger
numbers of conidia were trapped during a wet year in comparison to a dry
year (Gerstenberger, 1983). Free moisture or periods of 1002 relative
humidity are important in conidial production (Scharen, 1964),
liberation (Eyal, 1971) and dispersal (Faulkner and Calhoun, 1976) for
Septoria pathogens of wheat. Similar environmental effects are probably
active on spore production and release by musiva.
Conidia of ^ musiva have been trapped in increasing numbers from
June until mid-August in poplar plantations in Iowa (Young et al., 1980)
and Michigan (Gerstenberger, 1983). Periods of conidial release
potentially continue until final leaf fall, although this has not been
definitely established. Gerstenberger (1983) found that the dispersal
gradient for ^ musiva in plantations was steep and suggested that the
spores were dispersed in droplets of rain. Steep dispersal gradients
are typical of fungi which produce their spores in a slime matrix and subsequently require water to disperse the spores (Gregory et al, 1959; 78
Gregory, 1968). Other dispersal mechanisms may also be possible for S.
musiva because aerial dispersal of normally water-dispersed spores has
been documented in other Septoria species (Faulkner and Calhoun, 1976)
and for fungal pathogens with conidia of similar size and shape to S.
musiva such as Dothistroma pini Hul. (Gibson, et al., 1964) and
Brunchorstia pinea Hohn. (Luley and Hanion, 1984). Conidia of these
fungi may become airborne by a "splash take-off" method (Gregory, 1968;
Gregory et al., 1959; Gibson et al., 1964). Aerially dispersed conidia
of ^ musiva probably represent a small proportion of the conidia
released and may be insignificant in comparison to ascospores when both
spore types are present. For example, conidia of Leptosphaeria nodorum
(Septoria nodorum anamorph) can become airborne (Faulkner and Calhoun,
1976) and are the source of primary inoculum and spread of the pathogen
in areas where ascospores are not produced (Sanderson et al., 1985).
However, when ascospore inoculum is present, dispersal patterns and
frequent long distance spread of the fungus indicate ascospores are the
primary source of dispersal (Sanderson et al., 1985).
Foliar and Stem Infection
Inoculation of foliage and stems is usually conducted with conidia
produced in culture (Thompson, 1941; Zalasky, 1978; Long et al., 1983).
Leaf spot symptoms appear 10-20 days after inoculation (Thompson, 1941;
Long et al., 1983). Foliar symptoms vary with the species of Populus and the age of leaf tissue infected (Thompson, 1941). Leaf spots are typically circular, irregular, or angular, 1 to 15 mm in diameter. 79
reddish-brown to dark brown and have white to gray centers (Thompson,
1941). Leaves of some Populus species may have numerous small, angular
lesions or few, large (25 mm or greater), irregular spreading lesions
with white or gray centers that cover much of the leaf surface.
Under field conditions, leaf spot symptoms usually precede stem
infection symptoms in the spring (Bier, 1939) and sequentially in the
life of a stand (Waterman, 1954). Infection of stems occurs in the
current year's growth (Bier, 1939) before bark or rhytidome formation takes place (Zalasky, 1978). Major infection courts are wounds, stipules, buds, leaf petioles (Bier, 1939; Long et al.,1983) and apparently to a lesser degree lenticels (Long et al., 1983). Initial symptoms of stem infection appear in mid-June as dead leaves on leaders or axillary branches (Bier, 1939). Stem lesions are found at the bases of such dead leaves. Diseased stem tissue is usually black with lighter-colored areas in the center of the canker in which pycnidia may be found (Bier, 1939). Mycosphaerella populorum may girdle stems the first year of infection or continue to invade branch tissue and form a perennial canker (Bier, 1939; Filer, 1976). Perennial cankers have depressed centers and considerable callus tissue around canker margins
(Bier, 1939; Waterman, 1954). Because cankers cause bark to crack and peel away from the stem, infected branches are susceptible to invasion by secondary pathogenic fungi. Secondary pathogens such as Cytospora,
Dothichiza, Phomopsis. Fusarium, Botryodiplodia and wood decay fungi are frequently found fruiting and are isolated from the margins of second year and older Septoria cankers (Bier, 1939; Waterman, 1954; Morris et 80
al., 1975). These fungi are considered as secondary in the ecological
succession of fungi in stem tissue. Singly or collectively these
secondary pathogens can cause stem mortality (Morris et al., 1975).
Yearly expansion of cankers by ^ musiva and other fungi also weakens
stems and increases incidence of wind breakage (Bier, 1939).
Factors Affecting Disease Intensification During the Growing Season
Buildup of Septoria leaf spot and canker in plantations is related to several factors. Waterman (1954) noted that dense plantations are often the most heavily infected. Moore et al. (1982) and McNabb et al.
(1982) found a similar high level of disease incidence in dense coppiced stands. Dense stands develop a moist microenvironment by retarding evaporation (Scott et al., 1985). Extended periods of free moisture or high relative humidity apparently contribute to disease buildup.
Successful inoculation of stems and leaves of Populus has been conducted under extended periods of free moisture or high relative humidity.
Thompson (1941), Zalasky (1978) and Filer et al. (1971) infected stems and leaves with 48 hour and longer post inoculation foliar and stem wetness periods.
Specific environmental conditions necessary for infection of stems and leaves by M^ populorum remain to be quantified. Effects of environment and interactions of stand characteristic with environmental conditions have been a recent topic of increased study for Septoria diseases of wheat. Initial experiments showed that a two hour post inoculation dew period was sufficient to cause infection of highly 81
susceptible cultivars of wheat by Leptosphaeria nodorum (Bronnimann et al., 1972). Longer periods of 72 to 96 hours are usually considered optimal for maximum infection of wheat (Tomerlin, 1985). Infection levels and disease severity also vary considerably with temperature
(Tomerlin, 1985) and wheat cultivar (Shaner and Finney, 1982).
Both microclimatic factors associated with wheat canopy structure and general weather patterns that contribute to extended periods of moisture affect the severity of Septoria diseases of wheat. Scott et al. (1985) found that less disease developed in the lower canopy densities associated with tall varieties than in the more dense canopies of the dwarf wheat varieties. This effect was partially attributed to reduced leaf surface wetness on tall varieties in comparison with short statured wheats. Less dew formed and dew periods were shorter in duration because of the more open canopies of the taller varieties. The authors concluded that these conditions were sufficient to reduce disease severity in this study. A similar increase in rust, caused by
Melampsora medusae Thum., in denser Populus stands was quantified by
Schipper (1976). Greater infection by the rust fungus in the dense stands was attributed to longer periods of free moisture from dew, rain or irrigation, although this effect was not quantified.
Canopy density also may influence disease development by affecting spore dispersal and infection courts. For example, in dense stands of wheat, vertical "laddering" of splashed-dispersed conidia on overlapping leaves has been shown to increase disease incidence in shorter wheat varieties (Bahat et al., 1980). Spore dispersal as well as number and 82
availability of infection courts in the Mycosphaerella-Populus system
may also be affected by stand density. Bowersox and Merrill (1976)
correlated a decrease in canker severity with decreasing growing space
per tree. In retrospect, it appeared that increased stand density
decreased the number of available infection courts. A relationship
between stand density, infection and canker incidence has been shown for
Hypoxylon canker of aspen. In the Hypoxylon-aspen system, fewer side
branches, (the main infection court for Hypoxylon mammatum (Wahl.)
Mill.), develop under greater stand densities. Reduction in the number
of infection courts results in decreased disease incidence with
increased stand stocking (Anderson, 1964; Anderson and Anderson, 1968).
Clearly, the relationship between stand density and disease incidence is
complex and may require close study in each disease system to determine
the importance of various factors that affect disease intensification.
Because all aspects of the infection cycle of Septoria diseases of
wheat require moisture, regional weather patterns directly influence the
severity of epidemics. Septoria diseases of wheat are usually only a
problem in abnormally wet years (Shaner and Finney, 1976). Shaner and
Finney (1976) related "very severe" and "severe" epidemics of Septoria
tritici blotch to years with frequent rains in a 40 day period prior to
harvest of spring wheat. In a second study, disease severity was
negatively correlated with days without precipitation, temperatures
below 7 C and seven other meteorological variables that measured
moisture and temperature related parameters (Coakley et al., 1985).
Since the effect of moisture and other factors on Septoria diseases of 83
wheat is complex (Tomerlin, 1985), attributing epidemics to a single
environmental or pathogen related factor is seldom possible. A similar effect of a range of environmental factors on the various aspects of the secondary disease cycle of ^ musiva may exist. 84
MATERIALS AND METHODS
Field Experiments and General Sampling Design
Field experiments to determine weekly conidia densities, foliar and
stem infection periods and environmental measurements were carried out
as described in Section 1. Data analysis and development of regression
equations also were conducted as in Section 1.
Greenhouse and Laboratory Experiments
General procedures
Isolates of Septoria musiva were grown on a V-8 juice medium of 200 ml of cleared V-8 juice, 2 g of calcium carbonate, 15 g agar and 800 ml distilled water. The V-8 juice was cleared by centrifugation at 10,000 rpm for 4 minutes. Differences were evident between lots of V-8 juice and an attempt was made to utilize lots that gave a consistent color after centrifugation.
Two isolates were compared throughout the study. One isolate, which was cultured from the edge of a two year old canker, was single spored after isolation. The second isolate was a single ascospore isolate from overwintered leaves. Both isolates exhibited a marked propensity to lose sporulation ability and to sector with serial transfer. This was avoided by maintaining isolates on poplar stems in the greenhouse, storing a spore suspension of each isolate in sterile glycerol at -75 C, and transfering cultures by single or multi-spore isolation and selecting for the original culture type. Conidia for 85
inoculation and germination experiments were obtained by growing
cultures under continuous florescent light for 1.5 to 2.5 weeks at room
temperature, removing spores from freshly speculating colonies with a
sterile needle and dispersing the spores in sterile distilled water.
Foliar inoculations were made on the lower leaf surface of the
first 5 fully expanded leaves except in certain inoculum density studies
in which the tops of leaves were inoculated. Leaves were inoculated
with .05 ml of conidial suspension from a DeVilbiss number 31 glass atomizer (DeVilbiss Co., Toledo, Ohio) using a calibrated number of sprays.
Stem inoculations were made with a number 4 camel hair artists brush on the stems and lower petioles of unwounded branches.
Approximately .05 ml of the conidial suspension was painted on the stem and petioles between the five leaves selected for foliar inoculation.
Spore germination
Experiments on conidial germination, germ tube elongation and number of germ tubes per spore were conducted as in ascospore germination experiments (Section 1) except that a spore concentration of
1.25 X 105 was atomized on the surface of the water agar.
Relative humidity trials
Spores were germinated for 16 hours at various relative humidities maintained with glycerol-water mixtures (Johnson, 1940). Leaf discs cut from clone NC5272 were inoculated using the DeVilbiss atomizer. After 86
inoculation, the leaf discs were incubated at 100% relative humidity
where spores were kept wet continuously, and 100%, 97% and 92.8%
relative humidity with spores allowed to dry after delivery. A set of
spores also was germinated on water agar as a check. The experiment was
repeated over time and one hundred spores in each of four replicates
were evaluated for germination.
Inoculum density experiments
Foliage and stems on rooted potted cuttings were inoculated with a
spore suspension of narrow inoculum densities and placed in dew chambers
at 23 C for 24 hours. Two sets of experiments were conducted. In the
first set, the upper leaf surfaces and stems were inoculated with 500,
5000, 25000, 37500 or 50000 spores. Three rooted cuttings were
inoculated in each of four replicates which were repeated over time. In
the second experiment, the lower leaf surfaces were inoculated with 250,
1562, 3125, or 6250 spores per leaf. The experiments and two clones in each experiment were analyzed separately with analysis of variance.
Regression lines were fit where isolate differences were significant in the analysis of variance.
Time experiments
Foliage and stems on rooted potted cuttings of clone NC5272 were inoculated and placed in a dew chamber at 23 C for 0, 4, 8, 16 or 24 hours before removal and drying with a hand-held, blow drier. Three plants were inoculated in each of four replicates. 87
RESULTS
Field Experiments
Spore trapping
Conidia of Septoria musiva were first trapped in increasing numbers
JD 153 in 1984 and JD 135 in 1985 (Fig. 13 and 14). Low, non-increasing
levels of conidia (not detected at counts made at 400X but were found at
100X magnification) were trapped after JD 117 in 1984 and JD 114 in
1985. Differences in the time of peaks were evident between the vaseline spore trap (VST) and the monofilament trap (MST) (Fig. 13 and
14). Conidia were trapped with the VST in all weeks with measurable rainfall. The greatest number of spores detected with the VST occurred in weeks with rainfall amounts of 0.1-0.2 cm per week. In comparison, the MST trapped no or low numbers of spores in weeks with 0.15 cm per week or less of rainfall and generally the greatest number of spores were trapped in weeks with the highest rainfall (Fig. 13 and 14). Spore trapping was discontinued while leaves were still remaining on trees in
1984, however, spores were trapped by both spore trap methods until leaf drop in 1985.
Foliar and stem infection periods
Foliar infection was found in all sampling periods when measurable levels of conidia were trapped by the VST (Fig. 15). Foliar infection ratings during the period of conidial release were generally lower than those recorded during peak ascospore discharge periods (Fig. 11 and 15). Figure 13. Rainfall and spore trap data monitored in 7-day intervals during the period of conidial release in 1984. Vaseline spore trap data are an average of two slides at each of three sites and are expressed as the number of conidia per cm^ of trapping surface. Monofilament spore trap data are an average of two traps at each of three sites and are expressed as 1/100 of the total number of spores collected per trap 15 Rainfall 10 5 0
2000 r • MONOFILAMENT TRAP 1984 : O VASELINE TRAP 1500
1000
500 B-
0 Q"
150 175 200 225 250 275 JULIAN DATE Figure 14. Rainfall and spore trap data monitored in 7-day Intervals during the period of conidial release In 1985. Vaseline spore trap data are an average of two slides at each of three sites and are expressed as the number of conidia per cra^ of trapping surface. Monofilament spore trap data are an average of two traps at each of three sites and are expressed as 1/100 of the total number of sprores collected per trap T——r
Rainfall 8 4
0 Î.• 9 9^ ' ' ' ' ' ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' '
• MONOFILAMENT TRAP 1985 o VASELINE TRAP 1000
500
ja 0 - s%i @ i'Qta 0 B
' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I ' ' ' ' I . . . . 125 150 175 200 225 250 275 300 325 350 JULIAN DATE 1 o 1984
O 1985
,1 I I I I I I I : I I I I I I I I I I I I I I I I I I I I I I I I I 125 150 175 200 225 250 275 300 JULIAN DATE
Foliar Infection of clone NC5272 during the period of conidlal release in the 1984 and 1985 field seasons. Foliar infection ratings were based on a scale of 0-5 where 0 = no Infection and 5 = 75+ lesions per leaf 93
Stems of clone NC5271 were not infected in either 1984 or 1985
during the period of conidial release. Stem infection of clone NC5272
occurred throughout the growing season in both years (Table 11). In
relation to spore trap data, stems were infected during periods of mixed
ascospore and conidial populations and in periods when only conidia were
trapped.
Environment and disease
Correlation coefficients between weekly foliar and stem infection and environmental and spore trap data were determined (Tables 12-14).
Spore trap data were poorly correlated with foliar and stem infection and the environmental variables measured. The monofilament spore trap data were correlated with foliar ratings in 1985, and 1984 and 1985 data combined (Tables 13 and 14). The vaseline spore trap data were not correlated significantly with any disease ratings. The interaction between average temperature during a wetting period and hours of wetness
(longest and total hours wet for the week) were generally better correlated with foliar and stem infection than were duration of wetting period or average temperature during a wetting period alone.
Regression equations developed to relate disease levels to the environment and spore trap data generally accounted for less of the total variation than equations developed during the period of ascospore release (Tables 15-17). Interaction between average temperature during wetting and hours of wetness contributed most frequently to regressions on both foliar and stem infection. Rainfall, longest wetting in an 94
Table 11. Stem infection of clone NC5272 exposed in the field for 7 day periods during the period of conidial release in 1984 and 1985. Clone NC5271 was not infected during these exposure periods
1984 1985 Julian No. cankegs Julian No. cankers date^ per stem date per stem
153 0.00 142 0.06 160 0.00 149 0.00 167 0.33 156 0.20 174 0.26 163 0.06 181 0.00 170 0.66 188 0.00 177 0.00 195 0.00 184 0.00 202 0.33 191 0.06 209 0.00 198 0.20 216 0.00 205 0.33 223 0.00 212 0.00 230 0.00 226 0.20 240 0.00 233 0.20 247 0.13 240 0.13 254 0.33 247 0.00 261 0.00 254 0.00 267 0.00 262 0.60 274 0.00 269 0.20 276 0.20
^Exposure period ending on Julian date indicated.
^No more than one lesion per stem occurred on Clone NC5272 in any infection period, so number of cankers per stem = proportion stems infected. Table 12. Correlation coefficients between foliar and stem infection data and spore trap and environmental parameters measured during the period of conidial release in the field for 1984
Foliar No. cankers Vaseline Monofilament rating per stem spore trap spore trap NC5272
Vaseline spore trap 0.083 -0.184 Monofilament spore trap 0.284 0.201 0.098 - Longest wet^ 0.414 0.758*** -0.282 0.013 Total wet^ . 0.517* 0.585** -0.127 0.260 Temp longest wet 0.583** 0.174 0.282 0.324 Temp total wet 0.550** 0.170 -0.127 0,292 Temp X longest wet° 0.492* 0.834*** -0.144 0.145 Temp X total wet 0.573** 0.580** -0.079 0.340 Rain® 0.471 0.526 -0.403 -0.074
.Longest and total hours of wetness in a 7-day sampling period. Average temperature during total and longest wetting period. ^Interaction between longest period of wetness and average temperature during the wetting period. Interaction between total hours of wetness and average temperature during these wetting periods. Rainfall in centimeters per week. *Significant at P = .05. **Significant at P = .01. ***Significant at P = .001. Table 13. Correlation coefficients between foliar and stem infection data and spore trap and environmental parameters measured during the period of conidial release in the field for 1985
Foliar No. cankers Vaseline Monofilament rating per stem spore trap spore trap NC5272
Vaseline spore trap 0.296 -0.194 Monofilament spore trap 0.542*** -0.068 0.456* - Longest wet® 0.548** 0.151 0.188 0.520** Total wet . 0.622*** 0.368 0.135 0.514** Temp longest wet 0.289 0.001 0.188 0.079 Temp total wet 0.425* 0.115 •0.078 0,040 Temp X longest wet° 0.626*** 0.143 0.140 0.474** Temp X total wet 0.695*** 0.394* 0.108 0,394* Rain® 0.496** 0.096 0.209 0.600***
.Longest and total hours of wetness in a 7-day sampling period. Average temperature during total and longest wetting period. ^Interaction between longest period of wetness and average temperature during the wetting period. Interaction between total hours of wetness and average temperature during these wetting periods. Rainfall in centimeters per week. ^Significant at P = .05. **Significant at P = ,01. ***Signifioant at P = .001. Table 14. Correlation coefficients between foliar and stem infection data and spore trap and environmental parameters measured during the period of conidial release in the field for 1901 and 1985 combined
Foliar No. cankers Vaseline Monofilament rating per stem spore trap spore trap NC5272
Vaseline spore trap 0.126 -0.198 Monofilament spore trap 0.350* -0.028 0.211 - Longest wet® 0.516** 0.281 -0.076 0.222 Total wet® . 0.547** 0.355* 0.010 0.371** Temp longest wet 0.382*** 0.172 0.286 0.240 Temp total wet 0.151*** 0.100 -0.241 0.202 Temp X longest wet° 0.584*** 0.290 0.299 0.268 Temp X total wet 0.578*** 0.311* 0.268 0.382** Rain® 0.431** 0.167 -0.156 -0.182
.Longest and total hours of wetness in a 7-day sampling period. Average temperature during total and longest wetting period. "interaction between longest period of wetness and average temperature during the wetting period. Interaction between total hours of wetness and average temperature during these wetting periods. ^Rainfall in centimeters per week. ^Significant at P = .05. **Significant at P = .01. ***Significant at P = .001. Table 15. Regression models developed to relate foliar and stem infection levels to spore trap data and the environment during the period of oonidial release for 1981, All variables used in the model are significant at P = ,05
Dependent Intercept Variable? Variable2 R' variable (RC®) (RC)
Foliar -0,003 Temp longest wet Rain^ 10,7* 0.58 rating (0,060) (0,077)
Infections -0.08 Temp X longest wet^ 36.5* 0.69 per stem (0,001) NC5272
Regression coefficient,
^Average temperature during longest continuous wetting period for the week.
^Rainfall in centimeters per week.
^Interaction between longest continuous wetting period for the week and average temperature during the wetting period.
^Significant at P = ,001, Table 16, Regression models developed to relate foliar and stem infection levels to spore trap data and the environment during the period of conidial release for 1985. All variables used in the model are significant at P = .05
2 Dependent Intercept Variablel Variable2 Variables F variable (RC^) (RC) (RC)
Foliar 0.46 Temp X total wet^ Rain^ Mono spore 13.2* 0.66 rating trap (0.003) (-0.169) (0.00001)
Infections 0.01 Temp X total wet Temp X long wet® Rain 5.9** 0.47 per stem (0.002) (-0.002) (-0.482) NC5272
^Regression coefficient.
''interaction between total hours wetness and average temperature during the wetness period.
°Rain in centimeters per week.
'^Conidia collected in the monofilament spore trap,
^Interaction between the longest continuous wetting period and average temperature during the wetting period.
*Significant at P = .001.
^^Significant at P = .004. Table 17. Regression models developed to relate foliar and stem infection levels to spore trap data and the environment during the period of conidial release for 1984 and 1985 combined. All variables used in the model are significant at P = .05
2 Dependent Intercept Variablel Variables F R variable (RC)® (RC)
Foliar 0.002 Longest wet^ Temp longest wet° 14.39* 0.42 rating (0.037) (0.048)
Infections 0,03 Temp X total hrs wet^ - 5.2** 0.11 per stem (0.0003) NC5272
^Regression coefficient.
^Longest continuous wetting period for the week.
^Average temperature during the longest wetting period.
"^Interaction between total hours wetness and average temperature during the wetness periods,
*Significant at P = .001.
**Significant at P = .02, 101
exposure period and temperature during the longest wetting period were
used in a number of equations. Spore trap data (MST) were used in only
one of the models developed.
Laboratory Experiments
Spore germination
Conidial germination and germ tube elongation of the single
ascospore (SA) and single conidial (SC) isolates were similar to those
determined for ascospores from overwintered leaves, except both isolates
germinated at 35 C (Fig. 16A, 16B, 17A and 17B). Ascospores did not
germinate at this temperature. Germination of the SC isolate was 50 %
or greater at 25 and 30 C after 4 hours. Both isolates germinated
primarily from end cells of the conidium. Occasional germ tubes
developed from the center cell and rarely were more than three germ tubes per conidium observed after 16 hours. The number of germ tubes at each temperature-time combination is given in table 18.
Relative humidity and spore germination
Both the SA and SC isolates germinated better on water agar than at
100% relative humidity on leaf discs (Table 19). Allowing conidia to dry before placing in 100% relative humidity reduced germination by approximatley one-half. Conidia of both isolates germinated at 97% but did not germinate at 92.8% relative humidity. A CANKER ISOLATE B ASCOSPORE ISOLATE
A IOC 100 A 0 c O 5C 0 15 0 - O 20 C o 20C • 25C • 25 C § 80 # 30 C • 30C • 35 C 60
40 / 20
0 8 12 16 8 12 HOURS HOURS
Figures 16A and 16B. Percentage germination of a single conidium isolate from a canker and a single ascospore isolate from overwintered Pôpulus leaves at various temperature-time combinations. Conidia were germinated on water agar and each data point is an average of five replications of 125 spores per replication 1 1 : A CANKER ISOLATE B ASCOSPORE ISOLATE
A IOC 100 A IOC 0 15C 100 - 0 15C o 20 C o 20 C ; • 25 C • 25 C 1 75 - • 30 C / 75 - O 30C i • // w y — QQ 50 • / 50 -
o i 25 A 25 - to
• 1 .1, „ 0 à 8 12 16 0 8 12 16 HOURS HOURS
Figures 17A and 17B. Germ tube elongation of a single conidium isolate from a canker and a single ascospore isolate from overwintered Populus leaves at various temperature-time combinations. Conidia were germinated on water agar and each data point is an average of five replications of 125 spores per replication
1 104
Table 18. Average number of germ tubes per spore produced by a single ascospore (SA) and a single conidial (SO isolate at various hour-temperature combinations
SA
Temperature
Hour® 10 15 20 25 30 35
4 o o 0.0 1.0 1.0 1.1 0.0
8 0.0 1.0 1.7 1.8 1.9 1.0
16 1.0 1.2 1.7 2.0 2.0 1.4
SC
Temperature
Hour 10 15 20 25 30 35
4 0.0 0.0 1.0 1.1 1.1 0.0
8 1.0 1.1 1.6 1.8 1.9 1.0
16 1.0 1.3 1.6 2.0 2.1 1.0
^Hours incubation on 1,5% water agar. 105
Table 19. Germination of a single ascospore isolate (SA) and single conidial isolate (SC) at various relative humidities using glycerol-water mixtures
% Germination (Sem)® Treatment SA SC
1.5% water agar 95.0 ( 2.3) 99.0 (1.7)
100% wet^ 48.7 (10.4) 64.8 (8.1)
100% dry® 23.2 ( 9.2) 38.0 (3.9)
97% 6.0 ( 4.1) 11.7 (4.9)
92.8% 0.0 0.0
^Standard error of the mean.
^Conidia continuously wet during 100% relative humidity treatment.
^Conidia were allowed to dry before placement in 100% relative humidity treatment. 106
Inoculum density experiments
Bottoms of leaves were more susceptible to infection than the tops
of leaves of both NC5271 and NC5272 (Fig. ISA, 18B, 19A and 19B). No
significant difference in the number of lesions per leaf was found
between isolates when inoculations were made on tops and bottoms of
leaves of clone NC5272 (Fig. 18B, 19B, Tables 22 and 24). The SC
isolate caused significantly more lesions per leaf than SA isolate on
the tops and bottoms of leaves on clone NC5271 (Fig. 18A, 19A, Tables 21
and 23).
No stem infection of clone NC5272 occurred at 500 spores per stem.
The SC isolate caused significantly more stem lesions than SA on clone
NC5272 (Fig. 20A and Table 25), although no significant difference was found between isolates in percentage stems infected (Fig. 20B and Table
26). Clone NC5271 was not infected by SA at any of the inoculum densities used. The SC isolate infected this clone at 37,500 and 50,000 spores per stem. Eight percent and 16 percent of the stems were infected at these inoculum densities, respectively.
Effect of time on foliar and stem infection
Foliar and stem infection of clone NC5272 occurred after four hours in a dew chamber. A quadratic relationship was observed between foliar infection levels and time (Fig. 21A and Table 27). Stem infection was linear with time in these experiments (Fig. 21B and Table 28). 1 1 1 1 1 i 1 1 1 1 - A • . B / 0 6 - 0 ASCOSPORE ISOLATE / 20 T- Y=.02+.019X / o ASCOSPORE ISOLATE • CANKER ISOLATE / O/ • CANKER ISOLATE 5 Y=.24+.062X / Y=.07+.175X / 16 /o / 4 / 0 y/ / / 12 - - / 3 / • / / • / 8 y/O - 2 / — / O y/ 0 / •/ 4 1 - / - / 0 ^ — 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 >—1-1., I..,l 1—1 0 9LI—1—1.1 1 1 1—!.. 1, .1 • 1 • . . 1 . 1 1 1 1 1—1 . 0 20 40 60 80 100 0 20 40 60 80 100 CONIDIA/LEAF (X500) CONIDIA/LEAF (X500)
Figure ISA. Foliar infection of clone NC5271 at various inoculum densities inoculated on the tops of leaves. R-square = 0.54 for the ascospore isolate regression. R-square = 0.65 for the canker isolate regression
Figure 18B. Foliar infection of clone NC5272 at various inoculum densities inoculated on the tops of leaves. R-square =0.62 T 1 r 1 T 1 1 1 •• 1 1 D B o ASCOSPORE ISOLATE 20 / - 12 Y=-.66+.076X " 0 ASCOSPORE ISOLATE • CANKER ISOLATE / - o CANKER ISOLATE Y=.99+.109X / Y=.24+.176X 16 -
12 - /• / 0 8
-1 1 1 1 1 1 1 1 1 1 1 1 1 1 -iJ-j-i.iJ 1 1 1 1 0 20 40 60 80 100 120 0 20 40 60 80 100 120 CONIDIA/LEAF (X50) CONIDIA/LEAF (X50)
Figure 19A. Foliar infection of clone NC5271 at various Inoculum densities Inoculated on the bottoms of leaves. R-square =0.88 for the ascospore isolate regression and R-square = 0.71 for the canker isolate regression
Figure 19B. Foliar infection of clone NC5272 at various inoculum densities inoculated on the bottoms of leaves. R-square = 0.75 1 r T T r
o ASCOSPORE ISOLATE • 100 Y=-.01+.012X O ASCOSPORE ISOLATE • CANKER ISOLATE / • CANKER ISOLATE Y=-.14+.026X Y=4.84+.827X 80 S(/) / 60 / y / / o 40 / o / VO / 20 / D/ - / / I I 1,1 I I • 0 h ' ' ' * ' ' • I I ' • I • • ' I • ' • I I I I 20 40 60 80 100 0 20 40 60 80 100 CONIDIA/STEM (X500) CONIDIA/STEM (X500)
Figure 20A. Stem infection of clone NC5272 at various inoculum densities. R-square = 0.54 for the ascospore Isolate regression. R-square = 0.74 for the canker Isolate regression
Figure 20B. Percentage of stems infected of clone NC5272 at various inoculum densities. R-square = 0.67
I Y=.68-.26X+.029X Y=-.12+.049X 1 -
.8 -
6 -
.4 -
.2 -
0 8 12 16 20 24 8 12 16 20 24 HOURS WET HOURS WET
Figure 21A, Foliar infection of clone NC5272 after various time intervals in a dew chamber, R-square = 0.88
Figure 21B. Stem infection of clone NC5272 after various time intervals in a dew chamber. R-square = 0.81 m
DISCUSSION
Low non-increasing levels of Septoria musiva conidia were trapped
in April and May before conidial populations increased on a weekly
basis. These conidia were probably released from overwintered pycnidia
in leaves and cankers (McNabb et al., 1982; Waterman, 1954; Bier, 1939).
Levels of conidia monitored were probably too low to cause significant
levels of foliar and stem infection in comparison to increasing
populations of ascospores present in the same period. However,
overwintered asexual states of other ascomycete foliar and stem
pathogens can cause primary infection in the absence of the sexual state
(Pinon and Poissonnier, 1975; Sanderson and Hampton, 1978; Cook, 1974).
Weekly increase of conidial populations of ^ musiva commenced in early June in 1984 and late May in 1985. Conidial populations generally increased on a weekly basis during periods of wet weather in both years
(Fig. 13 and 14). High conidial populations were maintained throughout most of the summer and until final leaf drop in the fall when monitored in 1985.
Interpretation of peak conidial release periods depended on the spore trap method. The vaseline spore trap collected the highest conidial levels in weeks with less than 0.2 cm of rain. Peak periods of conidial release in 1984 would be interpreted as occurring in the latter part of the season (after JD 215) when low levels of rain fell (Fig.
13). In 1985, conidial peaks appeared to occur during weeks with low amounts of rainfall that were interspersed between wetter weeks (Fig. 112
14). In contrast, the monofilament spore trap collected few or no
spores in weeks with rainfall amounts of 0.15 or less. Peak conidial
populations in 1984 were found earlier in the season (before JD 215)
during weeks with high rainfall. Conidial levels then declined after JD
215 when decreased amounts of rain fell. In 1985, conidial populations
monitored by the monofilament spore trap generally increased during the
growing season with frequent rainfalls over 0.15 cm per week.
Interspersed in the general trend of increased conidial populations were
weeks with low amounts of rain when low conidial levels were monitored.
Efficiency of both spore traps and periods of peak spore collection
therefore varied depending on the level of rain. The hydrophobic nature
of the petroleum and washing of conidia off the spore trap surface after impaction apparently reduced the efficiency of the vaseline spore trap
in periods of high rainfall. The monofilament spore trap apparently requires moderate amounts of rainfall to wash conidia down into the collection vials and would therefore be inefficient in period with low amounts of rain. This may in part explain the low correlations between environmental parameters and spore trap data (Tables 12-14).
Foliage of clone NC5272 was infected in all weeks with measurable rain and when conidia were collected with the vaseline spore trap.
Foliar infection ratings often varied greatly with changes in weekly environmental and inoculum density conditions (Fig. 15). However, infection ratings were less correlated with weekly environmental and spore trap data than were foliar infection ratings made during the period of ascospore release. Regression models developed from the 113
period of conidial release also accounted for less of the total
variation than models developed during the period of ascospore
productivity.
Most regression models developed included a measure of temperature
during wetting periods and a measure of hours of wetness (total or
longest) or the interaction between temperature and wetness duration
(Tables 15-17). Rain and spore trap data were included in a few of the
models. Other environmental variables not measured also may have
influenced foliar infection. Coakely et al. (1985) found 9 météorologie
variables were correlated with the severity of Septoria tritici blotch
of wheat. Many of the variables pertained to weather conditions between
wetting or infection periods. Similarly, environmental factors during
and between wetting periods also affected, pycnidial and conidial
development (Scharen, 1964), conidial liberation (Eyal, 1971), release
and dispersal (Faulkner and Calhoun, 1976) and infection (Jeger et al.,
1981) in Septoria diseases of wheat. Germination of ^ musiva conidia
in this study also occurred at relative humidities below 100% (Table
19). Utilization of environmental variables that pertained only to
average temperature and duration of wetting periods resulting from rain
could account for the poorer correlations and regression models
developed for foliar infection during the period of conidial release.
In dew chambers, leaves of clone NC5272 were infected after 4 hours post inoculation wetness (Fig. 21A). Conidial germination was also
greater than 50% after 4 hours at or near optimal temperatures for the single conidial isolate from a canker (Fig. 16A). Low amounts of rain 114
released large numbers of conidia when monitored by the vaseline spore
trap (Fig. 13 and 14). Conidial release, germination, penetration of
stomata and infection of leaves could thus be expected even in weeks
with limited rain and short periods of foliar wetness. Susceptible
varieties of wheat were infected by ^ nodorum after 2 hours of post
inoculation wetness (Bronniman et al., 1972) and in 3 hour periods of
high relative humidity (Holmes and Calhoun, 1974). Longer periods of
wetness (72 to 96 hours) were required for maximum foliar infection of
wheat (Tomerlin, 1983). Foliar infection by S. musiva increased
quadratically with time in dew chambers (Fig. 21A). Longer periods of
wetness in the field probably were more important than brief wetting
periods in foliar disease intensification.
No stem infection of clone NC5271 occurred during the period of
conidial collection, although this clone was infected during periods of
peak ascospore productivity. Infection of clone NC5271 also was found
only at the two highest inoculum levels (single spore canker isolate)
used in laboratory experiments. Under the conditions of this study,
resistance present in NC5271 was sufficient to withstand infection under
the environmental and inoculum level conditions present during most of
the growing season.
Stem infection of NC5272 occurred over a range of environmental and
conidium levels in the field (Fig. 13, 14 and Table 11). In laboratory
experiments, stem infection levels were directly related to inoculum
density and period of post inoculation wetness in a dew chamber (Fig.
20A, 20B and 21B). Stem infection, however, was correlated poorly with 115
inoculum levels and environmental variables measured in the field. Stem
infection may be a complex process that was affected by factors other
than those monitored in this study. Relationships between spore
dispersal and infection courts may have been important. High inoculum
densities were required in laboratory experiments to cause significant
levels of stem infection of both clones (Fig. 20A and 20B). Dispersal
of conidia to infection courts at high levels may have been a relatively
rare event. Conidia are released in a slime flux (Waterman, 1954;
Thompson, 1941) and were collected only in weeks with measurable rain
(Fig. 13 and 14). Rain drops or splashed rain water containing high
levels of spores may have impacted on infection courts such as petioles,
lenticels and stipules (Long et al., 1983) at low frequencies. Washing of conidia down stems to these infection courts possibly was an
important mode of inoculation in the field (H. S. McNabb, Jr., personal communication). Rainfall intensity, duration and frequency may have affected splash dispersal and washing of conidia to infection courts more than the amount of rainfall quantified in this study. Closer research would be required to elucidate environmental effects on spore dispersal-infection courts relationships.
Dispersal of conidia to the foliage of Populus leaves also may have affected levels of foliar infection in the field. Upper and lower leaf surfaces differed greatly in susceptibility to infection by conidia
(Fig. 19A, 19B, 20A, and 20B ). Infection of Populus leaves by ascospores occurred through stomata (Niyo et al., 1985). Lower
(abaxial) leaf surfaces of both clones used in this study and most 116
Populus species contained the majority of stomata (Pallardy and
Kozlowski, 1979). Impaction of conidia on the lower leaf surface would
greatly increase the chance of successful penetration of stomata and
infection of foliage.
Differences between the single ascospore isolate from overwintered leaves and the single conidium isolate from a canker were evident in both foliar and stem inoculation experiments at various inoculum densities (Fig. 13-20). In foliar inoculations, isolate differences were not significant on the susceptible clone NC5272 but were significant on the more resistant clone NC5271 (Tables 21-24). The same general trend was present in stem inoculations. The ascospore isolate failed to infect clone NC5271 while infection by the canker isolate occurred at the two highest inoculum densities. On clone NC5272, a significant difference in the number of lesions per stem was found between isolates, however, the percentage of stems infected was not significantly different. Determination of virulence differences among isolates from different sources (overwintered leaves, stem cankers or leaf spots) or geographical areas should therefore be made on more resistant clones. Zalasky (1978) also found isolate-clonal interactions when inoculations were made on Populus deltoides and 2^ balsamifera seedlings from different geographical areas. Clonal-isolate interactions may partially explain the variation found in the resistance of different clones in different geographical areas (Ostry and McNabb,
1985). 117
LITERATURE CITED
Anderson, R. L. 1964. Hypoxylon canker impact on aspen. Phytopathology 54:253-257.
Anderson, G. W. and R. L. Anderson. 1968. The relationship between stand density of quaking aspen and incidence of hypoxylon canker. For. Sci. 14:107-112.
Anonymous. 1982. Brown spot. In J. B. Sinclair, ed. The compendium of soybean diseases. Am. Phytopathol. Soc. St. Paul, Minn.
Bahat, A., I. Gelernter, M. B. Brown and Z. Eyal. 1980. Factors affecting the vertical progression of Septoria leaf blotch in short statured wheats. Phytopathology 70:179-184.
Bier, J. E. 1939. Septoria canker of introduced and native poplars. Can. J. Res. 7:195-204.
Bowersox, T. W. and W. Merrill. 1976. Stand density and height increment affect incidence of Septoria canker in hybrid poplar. Plant Dis. Rep. 60:835-837.
Bronnimann, A., B. K. Sally and E. L. Sharp. 1972. Investigations on Septoria nodorum in spring wheat in Montana. Plant Dis. Rep. 56:188-191.
Butt, D. J. and D. J. Royle. 1979. Multiple regression analysis in the epidemiology of plant diseases. Pages 78-114. In J. Kranz, ed. Epidemics of Plant Diseases-Mathematical analysis and modeling. Springer-Verlag, New York.
Chupp, C. and A. F. Scherf. I960. Vegetable diseases and their control. John Wiley and Sons, New York.
Coakley, S. M,, L. R. McDaniel and G. Shaner. 1985. Model for predicting severity of Septoria tritici blotch on winter wheat. Phytopathology 75:1245-1251.
Cook, R. T. A. 1974. Pustules on wood as sources of inoculum in apple scab and their response to chemical treatments. Ann. Appl. Biol. 77:1-9.
Davis, J. J. 1915. Notes on parasitic fungi in Wisconsin. I. Wis. Acad. Sci. Art. Let. Trans. 18:78-92.
Eyal, Z. 1971. The kinetics of pycnospore release in Septoria 118
tritici. Can. J. Bot 49:1095-1099.
Faulkner, H. J. and J. Calhoun. 1976. Aerial dispersal of pycnidiospores of Leptosphaeria nodorum. Phytopathol. Z. 86:357-360.
Filer, T. H. 1976. Etiology, epidemiology and control of cankers in Cottonwood. Pages 226-233. In B. A. Theigles and S. B. Land, eds. Proc Symp. on eastern cottonwood and related species. Louisiana State University, Baton Rouge, Louisiana.
Filer* H., Ic McCracken, A= Mohn and W. K* Randall. 1971. Septoria canker on nursery stock of Populus deltoïdes. Plant Dis. Rep. 55:460-463.
Gerstenberger, P. E. 1983. Epidemiology of Septoria musiva (Mycosphaerella populorum) in intensive culture plantations of poplar. Unpublished M.S. Thesis. Iowa State University, Ames, Iowa.
Gibson, I. A. S., P. S. Christensen and F. N. Munga. 1964. First observations in Kenya on a foliage disease of pines caused by Dothistroma pini Mulberry. Comm. For. Rev. 43:31-48.
Gregory, P. H. 1968. Interpreting plant disease dispersal gradients. Ann. Rev. Phytopathol. 6:189-212.
Gregory, P. H., E. J. Guthrie and M. E. Bunce. 1959. Experiments on splash dispersal of fungus spores. J. Gen. Microbiol. 20:328-354.
Holmes, S. J. I. and J. Calhoun. 1974. Infection of wheat by Septoria nodorum and ^ tritici in relation to plant age, air temperature, and relative humidity. Tr. Br. Mycol. Soc. 63:329-338.
Jeger, M. J., E. Griffiths and D. G. Jones. 1981. Influence of environmental conditions on spore dispersal and infection by Septoria nodorum. Ann. Appl. Biol. 99:29-34.
Johnson, C. G. 1940. The maintenance of high atmospheric humidities for entomological work with glycerol-water mixtures. Ann. Appl. Biol. 27:295-299.
Kranz, J. 1979. The role and scope of mathematical analysis and modeling in epidemiology. Pages 7-54. In J. Kranz, ed. Epidemics of Plant Diseases-Mathematical Analysis and Modeling. Springer- Verlag, New York.
Long, R., T. W. Bowersox and W. Merrill. 1983. Incubation periods of 119
Septoria leaf spot and canker on inoculated Populus hybrids. Phytopathology 73:369-370. (Abstr.).
Luley, C. J. and P. D. Manion, 1984. Inoculum potential of Gremmeniella abietina in New York. Pages 86-95. In P. D. Manion, ed. Scleroderris canker of conifers. Nijhoff-Junk. Boston.
McNabb, H. S., Jr., M. E. Ostry, R. S. Sonnelitter and P. E. Gerstenberger. 1982. The effect and possible integrated management of Septoria musiva in intensive, short rotation culture of Populus in the north central states. Pages 51-58. In J. Zavtitovski and E. A. Hansen, eds. Proc N. Am. Poplar Counc. Meet. Kansas State University.
Moore, L. M., Ostry, M. E., L. E. Wilson, M. J. Morin and H. S. McNabb, Jr. 1982. Impact of Septoria canker, caused by Septoria musiva, on nursery stock and first year plantation culture. Pages 44-50. In J. Zavitkowski and E. A. Hansen, eds. Proc. N. Am. Poplar Counc. Meet. Kansas State University.
Morris, R. C., T. H. Filer, J. D. Solomon, F. I. McCracken, N. A. Overguard and M. J. Weiss. 1975. Insects and diseases of Cottonwood. USDA For. Serv. Gen. Tech. Rep. SO-8.
Pallardy, S. G. and T. T. Kozlowski. 1979. Frequency and length of stomata of 21 Populus clones. Can. J. Bot. 57:2519-2523.
Palmer, M. A., A. L. Schipper and M. E. Ostry. 1980. How to identify Septoria leaf spot and canker of poplar. N. C. For. Exp. Stn., St. Paul, Minn.
Peck, C. H. 1884. Ann. Rep. N. Y. St. Bot. 35:138.
Pinon, J. D. and M. Poissonnier. 1975. Etude 'epidemiologique du Marssonina brunnea (Ell. and Ev.) Magn. Eur. J. For. Pathol. 5:97-111.
Sanderson, F. R. and J. G. Hampton. 1978. Role of the perfect states in the epidemiology of the common Septoria diseases of wheat. New Z. J. Agric. Res. 21:277-281.
Sanderson, F. R., A. L. Scharen and F. R. Scott. 1985. Sources and importance of primary infection and identity of associated propagules. USDA Agric. Res. Serv. 12:57-64.
Scharen, A. L. 1964. Environmental influences on development of glume blotch of wheat. Phytopathology 54:580-581.
Schipper, A. L. 1976. Poplar plantation density influences foliage 120
disease. Pages 81-84. In Intensive plantation culture- Five years research. USDA For. Serv. Gen. Tech. Rep. NC-21.
Scott, P. R., P. vr. Benedikz, H. G. Jones and M. A. Ford. 1985. Some effects of canopy structure and microclimate on infection of short and tall wheats by Septoria nodorum. Plant Pathol. 34:578-593.
Shaner, G. and R. E. Finney. 1982. Resistance in soft red wheat to Mycosphaerella graminicola. Phytopathology 72:154-158.
Shaner, G. and R. E. Finney. 1976. Weather and epidemics of Septoria leaf blotch of wheat. Phytopathology 65:781-785.
Sutton, T. B. 1978. Role of conidia of Venturia inaequalis in the epidemiology of apple scab. In Proc. Apple and Pear Scab Workshop. N. Y. Geneva Agric. Exp. Stn. Spec. Rep. 28:6-9.
Thompson, G. E.. 1941. Leaf spot diseases of poplars caused by Septoria musiva and ^ populicola. Phytopathology 41:241-254.
Tomerlin, J. R. 1985. Preliminary studies on the effect of interrupted wet periods on infection of wheat by Septoria nodorum. USDA ARS 12:68-71.
Waterman, A. M. 1954. Septoria canker of poplars in the United States. USDA Agric. Cir. 947 24p.
Young, K. E., H. S. McNabb, Jr. and M. E. Ostry. 1980. Aerial pathogens in a central Iowa plantation. Proc. Iowa Acad. Sci. 87(1):Centerfold 6.
Zalasky, H. 1978. Stem and leaf infections caused by Septoria musiva and ^ populicola on poplar seedlings. Phytoprotection 59:43-50. 121
SECTION III. Df VITRO ASCOCARP PRODUCTION OF MÏCOSPHAERELLA POPULORUM 122
INTRODUCTION
Genetic studies on many plant pathogens are limited because only
the asexual state is produced under normal culture conditions.
Manipulation of the culture medium and growth conditions is usually
necessary to stimulate iri vitro production of sexual fruiting
structures. Cultures from ascospores or conidia of ^ populorum from
field material results in production of conidia under normal conditions
(Thompson, 1941). Production of spermagonia and ascocarps in vitro have
not been reported on the various media used to grow the fungus in
culture. The purpose of this study was to determine if ascocarps of M.
populorum could be produced ^ vitro and to define the basic conditions
for production. A preliminary report of this research has been made
(Luley, 1985). 123
LITERATURE REVIEW
Mycosphaerella Jonhs. is a large and cosmopolitan loculoascomycete
genus including both plant parasites and saprophytes. Ascocarps of
Mycosphaerella species can be found associated with the overwintered
plant parts of monocots, dicots, gymnosperms, mosses, ferns, and algae
(Barr, 1972). A few species produce their ascocarps on living plant
parts or in necrotic lesions on living leaves (Luttrell, 1973; Barr,
1972). The following review will focus on the general characteristics
of the genus Mycosphaerella, the Mycosphaerella species associated with
Populus in North America, and on the factors affecting in vitro ascocarp
production of species of Mycosphaerella and related genera.
Characteristics of the Genus Mycosphaerella
Mycosphaerella is classified within the subclass
Loculoascomycetidae, order Dothideales and the family Dothideaceae
(Alexopoulos and Mims, 1979; Luttrell, 1973; Barr, 1972). Other classification schemes which place the genus in the family
Mycosphaerellaceae have been suggested (Gaumann, 1964; Miller, 1949).
As a whole, the fungi in this genus should exhibit the following characteristics; (1) uni- or multiloculate perithecoid ascostroma
(pseudothecium); (2) bitunicate asci that forcibly discharge ascospores through an exposed ostiole; (3) aparaphysate locules formed by the development of asci from a basal bush into the locule; (4) ascospores biseriate, crowded or in a fascicle within the ascus and (5) ascospores 124
hyaline or dull brown, two celled and usually slightly constricted at
the septum. Niyo et al. (1986), after a light and electron microscope
study of populorum, concluded that the fungus conforms to the
characv^ristics outlined above and is well placed in this genus.
Barr (1972) indicated Mycosphaerella is "rich in species" though
synonymy is incomplete and the number of species is less than published
descriptions would suggest. Gaumann (1964) noted the genus contained
over 1000 species. These species can be separated into two subgenera,
Mycosphaerella and Didymellina (v.Hohnel) Barr., based on conidial
states and the shape of the ascus (Barr, 1972). The conidial states in
the Mycosphaerella are classified in at least eight different form
genera including Septoria Sacc., Lecanosticta Syd., Ramularia Unger.,
Cladosporium Link ex. Fr., Cercospora Fr., Passalora Fr., Polythrincium
Kunze ex. Fr., and Stigmina Sacc. (Barr, 1972). Alexopoulos ana Mims
(1979) noted that it is not possible to predict the imperfect state of an unidentified species of Mycosphaerella by inspecting the perfect state.
Many species of Mycosphaerella also produce spermagonia and spermatia that may be mistaken for microconidial states of these fungi
(Alexopoulos and Mims, 1979). Spermagonia are typically produced in the fall during the early stages of pseudothecium development (Higgins,
1936; Jenkins, 1939). Spermatia function as male cells in the transfer of nuclei during plasmogamy (Jenkins, 1939; Thompson, 1941; Higgins,
1936). 125
Mycosphaerella Species on Populus
At least eight Mycosphaerella species have been described from the
fallen leaves of Populus species in North America (Table 1). The
apparent synonymy and the absence of verifiable fruiting bodies and
ascospores in type material reduce the number to less than eight. The
majority of species are probably saprophytic and have not been
established as causing disease on poplars. Two species, ^ populorum
and 2^ populicola Thompson, and their respective conidial states, S.
musiva Peck and ^ populicola Peck, have been consistently reported as
poplar pathogens in North America (Thompson, 1941; Anonymous, I960).
Thompson (1941) described and separated these fungi on the basis of size. ^ populicola has consistently larger ascocarps, asci, and
ascospores, although the two species overlap (Thompson, 1941). Lower
cardinal temperatures for ^ vitro spore germination and growth of M. populicola, a more northern species with a more limited host range
(Thompson, 1941; Zalasky, 1978), may also help separate the two species.
M. populorum is well established as a foliar and stem pathogen on a range of Populus species (Thompson, 1941; Waterman, 1954). The original description of M. populicola (Thompson, 1941) and most subsequent reports (Anonymous, I960) suggest that the fungus is a leaf spot pathogen. However, Zalasky (1978) reported that both M. populicola and
M. populorum incited foliar and stem lesions on balsamifera seedlings after inoculations with conidia. Only populorum was reported as causing stem lesions on P^ deltoïdes, apparently a differential host for the two fungi (Zalasky, 1978). Future study on the pathogenicity of 125
populicola is probably warranted. ^ vitro mating studies may provide
additional insight on speciation and pathogenicity of these fungi.
Factors Affecting Ascocarp Production
Most Mycosphaerella species grow well vegetatively on artificial
media, but few produce ascocarps under normal cultural conditions
(Alexopolous and Mims, 1979). Manipulation of certain cultural
conditions has stimulated the production of the sexual stages and has
allowed further study of the factors that affect the sexual cycle.
Low temperatures are often needed to induce the formation of
ascocarps of Mycoophaerella species and other fungi. Barr (1958) found
that ascocarps of 2^ tassiana did not develop unless subjected to an
extended period of low temperatures. Similar results have been noted
for other Mycosphaerella species (Vaughan, 1916) and for related genera
(Wehmeyer, 1954; Keitt and Langford, 1941). Temperatures below 4 C slowed ascocarp development of inaequalis significantly (Keitt and
Langford, 1941) and below 0 C they may inhibit completely pseudothecial formation (Ross and Hamlin, 1962). Temperatures more conducive to vegetative growth usually result in reduced or inhibited development of sexual stages (Hawker, 1966). Ross and Hamlin (1962) and Keitt and
Langford (1941) found that no ascocarps of V. inaequalis developed above
15 C although the higher temperature supported abundant vegetative growth and sporulation. However, Barr (1958) found that M. typhae showed no low temperature stimulation of ascocarp development and ascocarps developed normally at room temperature. 127
Ascocarp formation of temperate ascomycetes typically occurs on
overwintered plant parts where exhaustion of nutrients may be a factor
inducing sexual reproduction (Hawker, 1966). Media employed to
stimulate sexual stages of these fungi in vitro are usually low in
nutrients, particularly carbohydrates (Hawker, 1966). Carbohydrate
concentrations in media used to stimulate ascocarp production of
Hycosphaerella and related species have been reported to range from 0,5%
to 2.5$ (w/v) of the culture medium (Barr, 1958; Keitt and Langford,
1941; Wehmyer, 1954). For example, Keitt and Langford (1941) produced mature ascocarps of V. inaequalis on media containing 0.1, 0.5, 1.0 and
1.5% malt extract but not on a medium with 2.5% extract. Ascocarp production was also most abundant at the two lowest levels of malt extract. Hawker and Chaudhuri (1947) also showed that sugar source as well as concentration is important in stimulation of ascocarp production.
Addition of sterilized plant parts or extracts of plant parts that support the fungus in the field may increase ascocarp production (Ross and Hamlin, 1962) by providing physical and chemical requirements not met by synthetic additions (Bezerra and Kimbrough, 1982). Other media supplements such as nitrogen and minor elements will affect the production of ascocarps (Ross, 1961). A review of the influence of various nutrients and environmental factors on sexual reproduction was made by Hawker (1966). She suggested that it was theoretically possible to control the type of growth and reproduction by manipulation of additions to media and the environment supporting growth. 128
The sexual compatibility of cultures will also determine the
success of in vitro ascocarp production. Sexual compatibility systems
reported in the ascomycetes are homothallism, secondary homothallism and
bipolar heterothallism with two alleles (Alexopolous and Mims, 1979).
Tetrapolar sexuality is only known from the basidiomycetes (Raper, 1953;
Fincham et al., 1979).
In vitro matings of single spore cultures are often used to
determine the sexual compatibility system present. Homothallic and
secondary homothallic fungi should produce ascocarps from single spore cultures (Korf, 1952), although the two systems differ significantly in genetic control of the process (Raper, 1966). Barr (1958) reported M. tassiana and typhae produced ascocarps from single spore cultures.
No attempt was made to differentiate the sexual compatibility system present.
Heterothallic species require two sexually compatible isolates for ascocarp formation to occur. Bipolar heterothallism with two alleles is very common among the ascomycetes (Raper, 1966). Keitt and Palmitter
(1938) showed Venturia inaequalis was heterothallic after ^ vitro and in vivo mating studies with eight single spore isolates of the fungus.
However, the "bewildering array" of variation and frequent anomalies in mating compatibility systems makes it difficult to generalize about the specific systems encountered with any one group of fungi (Raper, 1966) and should be considered when determining sexual compatibility systems. 129
MATERIALS AND METHODS
Ascospore Size Measurements
Overwintered Populus leaves were collected in the spring of 1984 and 1985 to evaluate ascospore size range of 2^ populorum. The leaves were microscopically examined for ascocarps of typical Mycosphaerella before inducing ascospore discharge on water agar. Spores were killed and stained after 15 minutes and measured at 400X with a Leitz microscope. Leaves were collected from mixed hybrid Populus plantations at the Iowa Conservation Commission nursery in Ames, 4-H camp near
Luther, Iowa, and Shimek State Forest in southeastern Iowa. Collections were also made from natural deltoides stands near Birmingham, and
Roland, Iowa and Hickory Grove State Park near Colo, Iowa.
Ascocarp Production ^ vitro
A poplar leaf decoction agar (PLDA) was made from 25 g overwintered naturally senescent leaves steamed in 250 ml distilled water for 30 minutes. The supernatant was added to 5 g malt extract, 15 g agar, and enough water to make a liter of medium. The medium was inoculated by streaking a mycelial mat containing spores across the agar surface.
Petri dishes were then sealed with parafilm and allowed to establish for two days at room temperature before placing in a cold room at 8 C in the dark. Four single ascospore isolates made from overwintered leaves and the single canker isolate described previously were crossed in all possible combinations. Crosses were made by removing spermatia 130
(produced on the same plates) produced in milky drops with a sterile needle and placing in sterile distilled water. Receiving plates were flooded with 1-2 ml of the spermatia suspension. Checks were uninoculated PLDA plates. Plates were checked periodically for developing ascocarps and ascospores were measured when mature ascocarps were found. Ascocarps were fixed in gluteraldyhde-formalin-acetic acid, embedded in parafin and sectioned at 8-15 microns with a rotary microtome. Sections were treated with conventional microtechnique procedures and stained in hemalum, safranin and cholorzol black E or fast green and safranin (Johansen, 1940) 131
RESULTS
Ascocarp Production ^ vitro
Spermagonia with spermatia were produced two weeks after
inoculation on poplar leaf decoction agar and placement in a cold room
at 8 C. Spermagonia were round to flask shaped (Fig. 24 and 25) and
contained rod-shaped spermatia that measured 4-5 X 1 jum. Spermatia were
produced in a milky white drop that oozed from spermagonia (Fig. 24).
Ascocarps with mature asci and ascospores developed two to three
months after cultures were crossed using spermatia and incubated (Fig.
26). Ascocarps, asci and ascospores were morphologically similar to those collected, sectioned and observed from overwintered Populus leaves
(Fig. 30-33). A thick layer of stromatic tissue occasionally surrounded the centrum (Fig. 27 and 32). Asci developed in a basal bush (Fig. 28) and each bitunicate ascus contained eight, irregularly biseriate ascospores (Fig. 30). Ascospore size measurements from culture were of similar size range to field collected material (Table 20).
Ascocarps were produced at low densities on plates where ascocarps developed. Crosses between the various single spore isolates often failed to produce ascocarps. Ascocarps were produced in a cross between a single ascospore isolate and a single conidium isolate from a canker.
No ascocarps were found on selfed plates. No conclusion on the sexual compatibility system was made because of the large number of unsuccessful matings. 132
Ascospore Size Measurements
Ascospore size ranges determined from overwintered leaves from six
locations in Iowa are presented in Table 20. A Mycosphaerella species
with ascospores that ranged from 6.7-9.2 X 2.0-2.6 jjm were not included in these measurements. Table 20, Ascospore size measurements of ascospores produced vitro and in overwintered leaves collected in various locations in Iowa
No. of No. of Stand Location Source leaves spores Averages Range dev^
Ames Hybrid poplar 13 625 15.1 X 4.3ym 11.8-22.5 X 3.5-5.9pm 0.70
4-H Camp Hybrid poplar 5 300 14.3 X 4.2pm 11.8-17.7 X 3.5-5.9pm 0.25
Shimek State Hybrid poplar 15 100 14.2 X 4.3wm 11.8-18.9 X 3.5-3.9pm 0.79 Forest
Hickory Grove P, deltoides 5 100 15.1 X 4.0um 11.8-18.9 X 3.5-3.9pm 0.89
Birmingham P, deltoides 13 875 14,5 X 4.2pm 11.8-21.3 X 3.5-4.7pm 0.42
Roland P, deltoides 5 125 15.3 X 3,9pm 13.0-18.9 X 3.5-4.7pm 0.51
Culture Crosses from - 125 17.6 X 4.4pm 11.8-24.8 X 2.4-5.9pm 0.95 single spore isolates
^Standard deviation of length measurements. 134
DISCUSSION
Spermagonia and ascocarps of Mycosphaerella populorum were produced
on a medium containing reduced carbohydrate levels, water soluble
extracts from overwintered leaves and when incubated at 8 C. Similar
conditions stimulated ascocarp production of other Mycosphaerella spp.
(Barr, 1958; Vaughan, 1916) and fungi in related genera (Keitt and
Langford, 1941). Ascocarps produced in culture were morphologically similar to field collected material (Fig. 30-33) and observations from other studies (Niyo et al., 1986; Thompson, 1941).
Ascospore measurements from culture also compared favorably with measurements made from field collections (Table 20) and other reports in the literature (Barr, 1972; Thompson, 1941). However, a smaller size range was included in the original description of ^ populorum by
Thompson (1941). Ascospore size ranges observed here and by Barr (1972) suggested that a wider range than originally reported were inclusive for
M. populorum.
The low density of ascocarps produced in each petri plate precluded determination of the sexual compatibility system of populorm.
Production of ascocarps on sterilized overwintered leaf discs (Ross and
Hamlin, 1962) or other defined media may increase the level of ascocarp production. Increased consistency of pseudothecial development under controlled conditions should allow elucidation of sexual compatibility systems and genetic determinants of pathogenicity. 135
LITERATURE CITED
Alexopoulos, C. J. and C. W. Miras. 1979. Introductory Mycology. 3rd ed. John Wiley and Sons, New York.
Anonymous. I960. Index of plant diseases in the United States. Agric. Res. Serv. Agric. Hand. No. 165.
Barr, M. E. 1972. Preliminary studies on the Dothideales in temperate North America. Contr. from the Univ. Mich. Herb. 9:523-638.
Barr, M. E. 1958. Life history studies on Mycosphaerella tassiana and Mycosphaerella typhae. Mycologia 50:501-513.
Bezerra, J. L. and J. W. Kimbrough. 1982. Culture and cytological development of Rhytidysterium rufulum on citrus. Can. J. Bot. 60:568-579.
Dennis, R. W. G. 1968. British ascomycetes. Second ed. J. Cramer, Lehre, Germany.
Ellis, J. B. and B. M. Everhart. 1892. The North American pyrenomycetes. Ellis and Everhart, Newfield, New Jersey.
Fincham, J. R. S., P. R. Day and A. Redford. 1979. Fungal genetics. Fourth ed. Blackwell Scientific Publ., Oxford.
Gaumann, E. A. 1964. Die Pilze. Birkhauser, Basel.
Hawker, L. E. 1966. Environmental influences on reproduction. Pages 435-469. In G. C. Ainsworth, and A. S. Sussman, eds. The Fungi. Vol II. Academic Press, New York.
Hawker, L. E. and S. D. Chaudhari. 1947. Growth and fruiting of certain ascomycetes as influenced by the nature and concentration of the carbohydrate in the medium. Ann. Bot. 9:185-194.
Higgins, B. B. 1936. Morphology and life history of some ascomycetes with special reference to the presence and function of spermatia. III. Am. J. Bot. 23:598-602.
House, H, D. 1920. An index to the New York species of Mycosphaerella. Ann, Rep. N. Y. State. Bot. 73:25-31.
Jenkins, W. A. 1939. The development of Mycosphaerella berkeleyii. J. Agric. Res. 58:617-620. 136
Johansen, D. A. 1940. Plant microtechnique. McGraw-Hill Book Co., New York.
Lilly, V. G. and H. L. Barnett. 1951. Physiology of the fungi. McGraw Hill, New York.
Luley, C. J. 1985. ^ vitro production of ascocarps of Mycosphaerella populorum. Proc. Iowa Acad. Sci. 92(5):Abstr. 40.
Luttrell, E. S. 1973. Loculascomycetes. Pages 135-219. In G. C. Ainsworth, F. K. Sparrow, and A. S. Sussman eds. The Fungi. Vol. rVA. Academic Press, New York.
Keitt, G. W. and M. H. Langford. 1941. Venturia inaequalis (Cke.) Wint. I. A groundwork for genetic studies. Am. J. Bot. 28:805-820.
Keitt, G. W. and D. H. Palmiter. 1938. Heterothallism and variability in Venturia inaequalis. Am. J. Bot. 25:338-345.
Korf, P. 1952. The terms homothallism and heterothallism. Nature 170:534-536.
Miller, J. H. 1949. A revision of the classification of the ascomycetes with special emphasis on the Pyrenomycetes. Mycologia 41:99-127.
Niyo, K, A., H. S. McNabb, Jr. and L. H. Tiffany. 1986. Ultrastructure of the ascocarps, asci and ascospore of Mycosphaerella populorum. Mycologia 78:202-212.
Raper, J. R. 1966. Life cycles, basic patterns of sexuality and sexual mechanisms. Pages 473-511. In G. C. Ainsworth, A. S. Sussman, eds. The Fungi. Vol. II. Academic Press, New York.
Raper, J. R. 1953- Tetrapolar sexuality. Q. Rev. Biol. 28:233-259.
Ross, R. G. 1961. The effect of certain elements, with emphasis on nitrogen, on the production of perithecia of Venturia inaequalis (Cke.) Wint. Can. J. Bot. 39:731-738.
Ross, R. G. and S. A. Hamlin. 1962. Production of perithecia of Venturia inaequalis (Cke.) Wint. on sterile apple leaf discs. Can. J. Bot. 40:629-635.
Saccardo, P. A. 1915. Notae mycologicae. Ann. Mycol. 13:115-146.
Thompson, G. E. 1941. Leaf spot diseases of poplars caused by Septoria musiva and ^ populicola. Phytopathology 31:241-254. 137
Vaughn, R. E. 1916. The development of Mycosphaerella pinoides in pure culture. Phytopathology 6:103. (Abstr.)
Waterman, A. M. 1954. Septoria canker of poplars in the United States. USDA Cir. No. 947.
Wehmeyer, L. E. 1954. Perithecial development in Pleospora trichostoma. Bot. Gaz. 115:297-310.
Zalasky, H. 1978. Stem and leaf spot infections caused by Septoria musiva and ^ populicola. Phytoprotection 59:43-50. 138
SUMMARY AND CONCLUSIONS
1. Ascospore productivity of Mycosphaerella populorum exhibited a
distinct seasonal periodicity. Ascospore production commenced in April
at approximately the time of bud swell on Populus deltoides and
continued for an extended period of 5-6 months. Peak ascospore
production occurred in late May in 1984 and early May in 1985.
2. Cumulative ascospore productivity was related to degree days,
base 0 C, when monitored in controlled temperature chambers and in the field. Ascospore productivity-degree day curves were skewed to the right and asymmetric about their inflection point. These curves were linearized with a Gompit transformation.
3. Ascospore productivity (0-982 of total productivity) was predicted using historic degree day-data and ascospore productivity curves developed from controlled temperature chambers. Significant deviations from predicted values occurred in the field during a dry year. Further research would be required to determine if the predictive system would be useful in other years and locations.
4. Ascospores of Mycosphaerella populorum were released primarily after rains. Dew was unimportant in the induction of ascospore release.
5. The majority of ascospores were trapped in daylight hours although wetting periods from rain and dew did not show a similar distribution.
6. Foliar infection of a susceptible clone (NC5272) occurred in all weeks ascospores were collected in the field with vaseline spore 139
traps. Foliar infection ratings were related to levels of ascospore inoculum monitored during the period of 0-98% of ascospore productivity.
7. Duration of wetting periods, average temperature during wetting periods and the interaction of these environmental variables were correlated with foliar infection ratings. Wetting periods that occurred between 0700-1900 hours, or the period when the majority of ascospores were collected, were generally the best correlated with foliar infection ratings.
8. Stem infection of clones NC5271 and NC5272 could be attributed solely to ascospores. Stems of clone NC5272 were infected during the period of peak ascospore production and during periods with mixed ascospore and conidial populations. Stem infection of clone NC5271 was found only during the period of peak ascospore production in the spring.
9. Levels of stem infection of both clones were correlated with duration of wetting periods, average temperature during wetting periods and the interaction of these environmental variables. Wetting periods that occurred between 0700-1900 were generally the best correlated with stem infection levels.
10. Results from this study indicated that environmental factors and ascospore inoculum levels conducive to stem infection of both clones occurred periodically during the period of 0-98% of total ascospore productivity.
11. Low, non-increasing levels of conidia were trapped in April and May. These conidia were probably released from overwintered pycnidia in leaves and stems. 140
12. Conidial populations increased on a weekly basis starting in
early June in 1984 and late May in 1985. Conidia were generally trapped
in increased numbers throughout the growing season and until leaves
dropped in 1985.
13. Interpretation of periods of peak conidial release depended on
the spore trap method. Vaseline coated slide traps collected the
majority of conidia in weeks of low amounts of rain when these weeks
were interspersed or follwed periods of high rain. Washing of the
vaseline surface and the hydrophobic nature of the vaseline jelly
limited the efficiency of this spore trap in periods of high rainfall.
In contrast, the monofilament spore trap collected the majority of conidia in weeks with high levels of rain. The monofilament spore trap
required higher rainfall than the vaseline spore trap to collect
measurable numbers of conidia.
14. Foliar infection of clone NC5272 occurred in all weeks measurable levels of conidia were collected with the vaseline spore trap. However, foliar infection ratings were poorly correlated with conidial levels monitored in the field.
15. Foliar infection ratings were correlated with the duration of wetting periods, average temperature during wetting periods and the interaction of these environmental variables. Relatively low correlations with the environmental variables monitored suggested that other environmental or biologic factors may have affected foliar infection.
16. Foliar infection of clone NC5272 occurred after a four hour 141
post inoculation period in a dew chamber. Longer periods of free
moisture were probably more important than brief wetting periods in leaf
disease build up in the field.
17- Stem infection of clone NC5272 was found in multiple weeks
throughout the period of conidial release. Clone NC5271 was not infected during the same period. Resistance present in clone NC5271 was apparently sufficient to withstand infection under most environmental and inoculum levels conditions in the field.
18. Stem infection levels were poorly correlated with conidial levels and environmental variables measured in the field. Environmental and biologic factors other than those monitored in the study may have also affected stem infection of plants exposed in the field.
19. The bottoms of leaves of the two clones used in this study were more susceptible to infection than the tops of leaves.
20. Significant differences in foliar and stem infection levels between a single ascospore isolate and a single conidial isolate from a canker were found on the more resistant clone NC5271. Isolate differences were generally non-significant on the susceptible clone
NC5272.
21. Ascocarps of Mycosphaerella populorum with mature asci and ascospores were produced iji vitro on a poplar leaf decoction agar after incubation in cold room. Ascocarp morphology and ascospore size measurements from culture were comparable to field collected material and reports in the literature.
22. The low frequency of ascocarp production vitro precluded 142
conclusion on a sexual compatibility system for ^ populorum.
23. The range of ascospore length measurements from field collected
material was wider than indicated in the original description of the fungus. U3
LITERATURE CITED
Bier, J. E. 1939. Septoria canker of introduced poplars and native, poplars. Can. J. Res. 17:195-204.
Bowersox, T. W. and W. Merrill. 1976, Stand density and height increment affect incidence of Septoria canker in hybrid poplar. Plant Dis. Rep. 60:835-837.
Cooper, D. T. and T. H. Filer. 1976. Resistance to Septoria leaf spot in eastern cottonwood. Plant Dis. Rep. 60:812-814.
Dinus, R. J. 1974. Knowledge about natural ecosystems as a guide to disease control in managed forests. Proc. Am, Phytopathol. Soc, 1:184-190,
Filer, T,H,, F, I, McCracken, C, A, Mohn and W. A. Randall, 1971, Septoria canker on nursery stock of Populus deltoïdes. Plant Dis. Rep. 55:460-463.
Gerstenberger, P, E, 1983. Epidemiology of Septoria musiva (Mycosphaerella populorum) in intensive culture plantations of poplar. Unpublished M. S. Thesis. Iowa State University. 74 pp.
Jones, A, L, 1978. Analysis of apple scab epidemics and attempts to improve disease prediction. Proc. Apple and Pear Scab Workshop. N, Y. Agric. Exp. Stn. Spec. Rep. 22:19-22,
Kranz, J, 1974. The role and scope of mathematical analysis and modeling. Pages 7-54. In J, Kranz, ed. Epidemics of plant diseases-Mathematical analysis and modeling. Springer-Verlag, New York.
McDonald, G. I., R. J. Hoff and W. R, Wykoff. 1981. Computer simulation of white pine blister rust epidemics. USDA For. Serv. Pap. INT-258,
McNabb, H, S., Jr,, M, E. Ostry, R. S. Sonnelitter and P. E. Gerstenberger. 1982. The effect and possible integrated management of Septoria musiva in intensive short rotation culture of Populus in the north central states. Pages 51-58. In J. Zavitovski and E, A, Hansen, eds. Proc. N. Am. Poplar Counc. Meet., Kansas State University.
Moore, L. M., M. E. Ostry, L. E. Wilson, M. J. Morin and McNabb, H, S., Jr. 1982. Impact of Septoria canker, caused by Septoria musiva, on nursery stock and first year plantation coppice. Pages 144
44-50. In J. Zavitkovski and E. A. Hansen, eds. Proc. N. Am. Poplar Counc. Meet., Kansas State University.
Ostry, M. E. and H. S. McNabb, Jr. 1985. Susceptibility of Populus species and hybrids to disease in the north central United States. Plant Dis. 69:755-757.
Schreiner, E. J. 1970. Mini-rotation forestry. U.S. For. Ser. Res. Pap. NE-174.
Stout, A. B. and E. J. Schreiner. 1933. Hybridizing poplars. J. Hered. 24:217-229.
Thompson, G. E. 1941. Leaf spot diseases of poplars caused by Septoria musiva and S^ populicola. Phytopathology 31:241-254.
Van Der Plank, J. E. 1963. Plant diseases: epidemics and control. Academic Press, New York.
Waterman, A. M. 1954. Septoria canker of poplars in the United States. USDA Circ. 947. 24p.
Waterman, A. M. and K. F. Aldrich. 1952. Surface sterilization of poplar cuttings. Plant Dis. Rep. 36:203-207.
Weiss, M. J., R. J. Collins, T. H. Filer and P. H. Peacher. 1976. Disease impact in cottonwood plantations. Pages 245-256. In B. A. Thielges and S. B. Land, eds. Proc. Symp. on Eastern Cottonwood and Related Species. Louisiana State University, Baton Rogue, Louisiana.
Zalasky, H. 1978. Stem and leaf infections caused by Septoria musiva and ^ populicola on poplar seedlings. Phytoprotection 59:43-50.
Zalasky, H., 0. K. Fenn, and C. H. Lindquist. 1968. Reaction of poplars to infection by Septoria musiva and Diplodia tumefaciens and to injury by frost in Manitoba and Saskatchewan. Plant Dis. Rep. 52:829-833. 145
APPENDIX: FIGURES AND TABLES 146
1 1 1 1 1 1 r
100
y 80 en C/î
% so
o o: k! 40
20
' ' ' I ' ' ' I ' ' ' I ' ' ' I ' ' ' I ' ' ' ' ' : ' 'I ' ' ' ' 100 120 140 160 180 200 220 240 260 JUUAN DATE
Figure 22. Percentage leaf tissue surface area remaining from leaf discs used in ascospore liberation tunnel experiments. Surface area was rated visually each week after discs were returned to the laboratory to collect ascospores 147
Table 21. Analysis of variance on the number of foliar lesions per leaf on clone NC5271 at various inoculum densities when inoculations were made on the tops of leaves
Source DF MS F
Block 3 8.4 6.3* Isolate 1 49.5 37.8** Density 4 24.1 Lin (1) 92.5 70.0** Quad (1) 1.1 0.8 LOF (2) 1.2 0.9
Isolate X Density 4 6.6 Isolate X Lin (1) 25.7 19.4** Isolate X Quad (1) 0.3 0.2 Isolate X LOF (2) 0.2 0.1
Error 27 1.3
*Significant at P = .02. **Significant at P = .001.
Table 22. Analysis of variance on the number of foliar lesions per leaf on clone NC5272 at various inoculum densities when inoculations were made on the tops of leaves
Source DF MS F
Block 3 58.9 2.3 Isolate 1 51.7 2.0 Density 4 457.6 Lin (1) 1754.6 67.2* Quad (1) 50.5 1.9 LOF (2) 12.7 0.4
Isolate X Density 4 11.2 Isolate X Lin (1) 43.9 1.6 Isolate X Quad (1) 0.6 0.0 Isolate X LOF (2) 0.1 0.0
Error 27 26.1
*Significant at P = .0001. 148
Table 23. Analysis of variance on the number of foliar lesions per leaf on clone NC5271 at various inoculum densities when inoculations were made on the bottoms of leaves
Source DF MS F
Block 3 14.2 2.5 Isolate 1 98.7 17.8** Density 3 185.2 Lin (1) 552.5 100.4** Quad (1) 3.0 0.5 LOF CD 0.1 0.02
Isolate X Density 3 11.4 Isolate X Lin CD 17.7 3.2* Isolate X Quad CD 15.6 2.8 Isolate X LOF CD 1.1 0.2
Error 21 5.5
^Significant at P = .05. **Significant at P = .001.
Table 24. Analysis of variance on the number of foliar lesions per leaf on clone NC5272 at various inoculum densities when inoculations were made on the bottoms of leaves
Source DF MS F
Block 3 27.1 1.0 Isolate 1 3.4 0.1 Density 3 672.7 Lin CD 1992.7 73.8* Quad CD 22.8 0.8 LOF CD 2.7 0.1 Isolate X Density 3 Isolate X Lin CD 0.3 0.01 Isolate X Quad CD 0.0 0.01 Isolate X LOF CD 0.4 0.02
Error 21 27.0
^Significant at P = .001. 149
Table 25. Analysis of variance on the number of stem lesions per stem on clone NC5272 at various inoculum densities
Source DF MS
Block 3 0.7 3.6* Isolate 1 3.0 14.4** Density 4 5.2 Lin (1) 19.8 94.2** Quad (1) 0.6 2.8 LOF (2) 0.1 0.5
Isolate X Density 4 0.9 Isolate X Lin (1) 2.9 13.8** Isolate X Quad (1) 0.3 1.4 Isolate X LOF (2) 0.2 1.2
Error 27 0.21
*Significant at P = .02. **Significant at P = .001.
Table 26. Analysis of variance on the percent of stems infected on clone NC5272 at various inoculum densities
Source DF MS
Block 3 2036.2 5.3* Isolate 1 680.0 1.7 Density 4 9894.0 Lin (1) 38863.2 101.7** Quad (1) 522.1 1.3 LOF (2) 190.8 0.5
Isolate X Density 414.5 Isolate X Lin (1) 28.1 0.1 Isolate X Quad (1) 1176.9 3.1 Isolate X LOF (2) 226.1 0.6
Error 27 382.0
*Significant at P = .005. **Significant at P = .001. 150
Table 27. Analysis of variance on the number of foliar lesions per leaf on clone NC5272 after various time intervals in a dew chamber
Source DF MS F
Block 3 11.3 2.2 Time 3 107.3 Lin (1) 298.7 58.5** Quad (1) 23.8 4.7* LOF (1) 0.01 0.01
Error 9 5.1
^Significant at P = .07.
**Significant at P = .01.
Table 28. Analysis of variance on the number of foliar lesions per stem on clone NC5272 after various time intervals in a dew chamber
Source DF MS F
Block 3 0.01 0.1 Time 3 0.7 Lin (1) 2.3 39.2* Quad (1) 0.01 0.1 LOF (1) . 0.01 0.1
Error 9 0.05
^Significant at P = .001. Figure 23. Ascospore liberation tunnel described by Hirst and Stedman (1962) used to collect ascospores from overwintered Populus leaves in field and laboratory experiments
Figure 24. Spermagonia produced on a poplar leaf decoction agar. Note spennatia oozing from spermagonia (Size bar = 5mm)
Figure 25. Cross section of a spermagonium produced in culture. Note spermatia at the top of spermagonium (Size bar = 15um) 152 Figure 26. Ascocarps produced on the surface of a poplar leaf decoction agar after incubation for three months at 8 C (Size bar = 500 um)
Figure 27. Young ascocarp produced in culture. Note immature asci and thick stromatic tissue surrounding the centrum (Size bar = 15 um)
Figure 28. Basal bush of asci developing in an immature ascocarp produced in culture (Size bar = 15 um)
Figure 29. Low magnification of a mature ascocarp of ^ populorum produced in culture (Size bar = 40 um) 154
% Figures 30-33. Mature ascocarps produced in culture on a poplar leaf decoction agar (Size bar = 15 urn)
Figure 33. Ascocarp of ^ populorum collected and section from an over wintered Populus leaf in the field (Size bar = 15 um) 156 157
ACKNOWLEDGMENTS
I would like to thank the members of my committee for their time
and help, Drs. R. F. Nyvall, D. C. McGee, and M. D. K. Owen for support
at various times. Dr. M. L. Gleason for his editorial suggestions. Dr.
N. R. Lersten for use of the microtechnique facilities, B. L. Wagner and
Dr. H. T. Horner for photographic help and Dr. H. S. McNabb, Jr. for his
assistance in the final phase of this work. I am particularly grateful
to Dr. L. E. Sweets for her guidance and incredible patience in the
Plant Disease Clinic and continual motivation throughout my stay at Iowa
State. Finally, to the Luley clan, and especially to my parents, for their understanding of the idiosyncrasies of a professional student.
hi mar