The Effect of Modifying the Microenvironment Among Geranium Stock Plants Within a Greenhouse on Botrytis Blight and Sporulation of Botrytis Cinerea 1992 Proposal
THE EFFECT OF MODIFYING THE MICROENVIRONMENT AMONG GERANIUM
STOCK PLANTS WITHIN A GREENHOUSE ON BOTRYTIS BLIGHT AND SPORULATION OF
BOTRYTIS CINEREA
INTRODUCTION Tetraploid (2N=36) and selected cultivars of diploid (2N=18)
geraniums (Pelargonium x hortorum L.H. Bailey) are propagated asexually
by cuttings. Stem blight caused by Botrytis cinerea Pers. ex. Fr. typically
begins in the broken or cut-off stem surface of the stock plant and progresses
downward, causing a dieback of the entire stem and in severe cases extends
into the base of the plant resulting in death (Nichols and Nelson, 1982).
Observations suggest that stem blight is a limiting factor in production.
Stock plants are pinched at regular time intervals or treated with the
growth regulator ethephon (Florel; 2-chloroethyl phosphonic acid) (Tjia
and Kim, 1975) to increase plant branching and the number of growing points
that can be removed as cuttings. This management practice produces low,
compact plants with dense canopies that limit light and air penetration
and promote senescence of the lower leaves (Rogers, 1982). Close spacing
of stock plants to maximize cutting production greatly enhances these conditions.
Under specific environmental conditions, Botrytis readily infects these
senescent leaves and sporulates, providing ample inoculum to infect stems
wounded during the harvest of cuttings.
Traditional methods of controlling stem blight include fungicide application
and sanitation. However, fungicide efficacy may be limited by a dense stock
plant canopy shielding senescent, sporulating stems and leaves from adequate
fungicide coverage. Also, the precise time that fungicides should be applied
to maximize efficacy is unknown. Observations from a commercial greenhouse
indicated that atmospheric conidial concentrations continued to increase
following calendar applications of chlorothalonil and iprodione to geranium
stock plants. Also, the occurrence of fungicide resistance remains a constant
threat (Dennis and Davis, 1979, Katan, 1982; Maude, 1980; Northover and
Matteoni, 1986).
Sanitation measures typically include the removal and destruction of
diseased plant material. Dead leaves at the base of plants and organic
matter in and under benches may support Botrytis growth. Melchers (1926)
isolated Botrytis (considered to be B. cinerea Pers.) from organic matter
in and on the sand of cutting benches, moist soils and elsewhere, that
may have potentially served as a source of inoculum. However, maintaining
the high standard of hygiene necessary to reduce inoculum sources is time-consuming
and costly for commercial geranium growers. The benefit of such sanitation
efforts has been questioned by Plant and Berger (1981) who concluded from
studies of B. cinerea on begonia that sanitation measures may be less effective
that previously theorized. Low initial disease was apparently compensated
for by an accelerated rate of disease development.
Cultural methods of controlling diseases caused by B. cinerea have not
been thoroughly investigated. Heursel and Kamoen showed that lignified
(“hard”) cuttings of Rhododendron spp. can be better stored without loss
due to B. cinerea than “soft” cuttings. Similarly, Cline and Neely showed
that when wounded geranium cuttings were allowed to “heal” prior to inoculation
with Pythium ultimum, disease incidence and severity decreased.
Controlling disease caused by B. cinerea through modification of the
greenhouse environment is an attractive addition to traditional control
methods. Environmental modification to control disease caused by B. cinerea
is typically aimed at enhancing air circulation and minimizing the duration
of free moisture on the plants (Rogers, 1982). B. cinerea is dependent
on a water film for couidial germination and infection, therefore, preventing
temperatures from reaching the dew point is an effective mechanism of disease
escape (Jarvis, 1989). Although event- driven, interactive computers are
available with the capacity for predicting disease epidemics and altering
the greenhouse environment, the data base necessary for the development
of software that will provide predictions with a high confidence level
is lacking.
BACKGROUND INFORMATION
The Epidemiology of Botrytis cinerea Among Geraniums within a Greenhouse
was the title of the principle investigator’s dissertation. In one portion
of this investigation, the effect of modifying the microenvivonment among
geranium stock plants within a research and commercial greenhouse on Botrytis
blight and sporulation of B. cinerea was investigated. The following results
were used as a basis for the proposed research. Combinations of plastic
mulch and intervals of forced heated air were incorporated among geranium
(Pelargonium x hortorum) stock plants within a research greenhouse. According
to the area under the incidence of disease progress curve (AUDPC) data,
the incidence of sporulating B. cinerea on necrotic lower leaves of mature
stock plants was significantly decreased in comparison to the control for
the following treatments; (1) white plastic on top of the pots, (2) intervals
of forced heated air into the plant canopy during 2200 to 0600 hours and,
(3) combination of plastic mulch and forced heated air. The AUDPC data
indicated that the combination of plastic mulch and forced heated air was
significantly more effective in reducing the incidence of sporulating B.
cinerea on the lower necrotic leaves of stock plants than the individual
treatments. Forcing heated air among stock plants was significantly more
effective in reducing the incidence of sporulating B. cinerea on the lower
necrotic leaves of stock plants than the plastic mulch. Fresh and dry weights
of necrotic leaves with sporulating B. cinerea collected from plants at
the end of the experiments indicated that the combination of plastic mulch
and forced heated air was most effective in reducing the incidence of necrotic
leaves with sporulating B. cinerea. According to this data, forced heated
air among stock plants was effective in reducing the incidence of sporulating
B. cinerea on necrotic leaves in comparison to the plastic mulch treatment
and the controls.
In addition, continuous, forced heated air applied from beneath an open-bottom
bench by an electric heater, fan, and poly tube was incorporated among
geranium stock plants within a commercial greenhouse. According to AUDPC
data, the continuous forced heated air significantly reduced the incidence
of stem blight and the incidence of sporulating B. cinerea on blighted
stems and necrotic leaves of stock plants in comparison to the control.
Fresh and dry weights of necrotic leaves of stock plants in comparison
to the control. Fresh and dry weights of necrotic leaves with sporulating
B. cinerea collected from plants at the end of the growing season indicated
that continuous forced heated air was effective in reducing the incidence
of necrotic leaves with sporulating B. cinerea in comparison to the control.
In addition, during days when grower activity was documented, B. cinerea
conidial concentrations were estimated using a Burkard recording spore
trap within the modified area were lower than the concentrations trapped
within the control area.
In summary, forced heated air reduced the incidence of stem blight and
the sporulation of B. cinerea on necrotic leaves and blighted stems among
geramum stock plants within a research and commercial greenhouse. In addition,
in a commercial greenhouse during days when grower activity was documented,
estimated B. cinerea conidial concentrations within the growing area with
forced heated air were lower than the concentrations trapped within the
control area.
Another study included in the principle investigator’s dissertation,
involved the influence of a microenvironment with low relative humidity
on stem blight of geranium caused by B. cinerea. The following information
from this study was used in preparing this research proposal: All stems
of geranium stock plants inoculated with B. cinerea and incubated in a
dew chamber within 12 hr of excising cuttings (stem wounding) became blighted.
Disease incidence maxima were 38 and 29% when plants were inoculated 24
hour and 3 days, respectively, following stem wounding and placement in
an environment of low relative humidity (RH<60%) prior to incubation
in a dew chamber. The area under the incidence of stem blight disease progress
curve (AUDPQ revealed a minimum of 24 hr in an environment of low RH between
stem wounding and subsequent inoculation and incubation in a dew chamber
was necessary to significantly limit stem blight incidence.
Stem blight occurred in a minimum average of 94% of the stems when plants
were exposed to an environment of low RH for 24 hr following inoculation,
prior to incubation in a dew chamber. When plants were inoculated and subjected
to an environment of low RH for 3,5, and 7 days prior to incubation in
a dew chamber, an average of 69, 85, and 50% of the stems, respectively,
became blighted. According to the AUDPC data, when inoculated plants were
placed in an environment of low RH for a minimum of 24 hr prior to incubation
in a dew chamber, stem blight incidence was significantly limited. The
AUDPC data indicated that the longer inoculated plants were maintained
in an environment of low RH prior to incubation in a dew chamber, the lower
the stem blight incidence.
In summary, a minimum lag time of 24 hr in an environment of low RH
between stem wounding and subsequent inoculation with B. cinerea conidia
decreased stem blight incidence. Atmospheric couidial concentrations may
be reduced by preventing sporulation on infected stems and leaves and minimizing
grower activity within the greenhouse immediately before and after stem
wounding. Results also suggest that an environment of low RH immediately
following stem wounding may limit stem blight incidence. A lower level
of stem blight incidence occurred when inoculated plants were placed in
an environment of low RH for a minimum of 24 hr prior to incubation in
a dew chamber. Results from studies conducted thus far within year 1 of
this proposal under objective 1 were also used in preparing this research
proposal:
The objectives were to determine the effect of length of dew period
at various temperatures on conidial germination and subsequent infection
and lesion production on geranium leaves and wounded geranium stems exposed
to a standardized number of conidia under controlled environmental conditions,
with the conidia applied in a dry state similar to that occurring in nature;
and to determine the effect of extended dew duration on postinfection hyphal
development within lesions.
An isolate of B. cinerea originally isolated from Michigan-grown geraniums
was used in all experiments because it was highly virulent and it sporulated
prolifically on artificial media. The fungus was grown on potato dextrose
agar (PDA) for 12-16 days at room temperature and under a 16 hour fluorescent
light photoperiod. A galvanized sheet metal cylinder (10″ in diameter by
20″ deep) was used as a settling tower. Conidia were collected from sporulating
cultures using a pasteur pipet connected to a water aspirator. At very
low suction conidia were collected in the pipet without being sucked into
the aspirator. Conidia were tapped out of the pipet onto weighing paper.
Inoculum was quantified by weight. Amounts of conidia weighing 1, 2, 3,
4, 5, 6, 7, 8, 9, and 10 mg were used as inoculum, and determined with
a hemacytometer to represent approximately 2.8 x and 49.0 x 10^6 conidia
for weights of 1 and 10 mg conidia, respectively. Over this range, each
1 mg of inoculum contained approximately 5.1 x 10^6 conidia.
For inoculation, leaf discs placed on moist filter paper within petri
dishes were positioned within the settling tower and dry conidia were dispersed
near the top of the chamber by directing a low velocity stream of air from
a pipet tip over the conidia on a piece of weighing paper. A cover was
positioned over the top of the cylinder to reduce air currents and allow
the conidia to settle on the leaf discs.
On wet geranium leaf discs, conidia of B. cinerea began to germinate
within 60 minutes of inoculation at the optimum temperature of 20 C. Four
hours after inoculation, 40% of the conidia had germinated and by 6 hours
77% of the conidia had germinated. This information is critical because
it represents what would occur in a grower situation on plant tissue and
provides the groundwork for a predictive system.
Cuttings were excised from stock plants 2 cm above a plant node using
flame sterilized razor blades. Dry B. cinerea conidia were used as inoculum.
Dry conidia were dusted onto the cut surface with a camel’s hair brush.
Control and inoculated plants were immediately placed into a darkened,
20 C commercial dew chamber. The prepared stubs were examined with an ISI-60
scanning electron microscope (International Scientific Instruments, Holland,
NY) operating at an accelerating voltage of 10kV. Representative photomicrographs
of inoculated and longitudinal surfaces were taken with a Polaroid 545
land camera with type 52 film (ISO 400) and a 35 mm Asaki Pentax using
TMAX 100 film. In addition, a stem from each treatment time was transversely
cut 5 mm from the inoculated surface and then cut longitudinally in half.
All cuts were made at the time of sampling with sharp, sterile scalpels.
Germination of the conidia and penetration of the wounde surfaced of
the stem occurred within 3 hours of inoculation. Enzymatic degradation
at the point of penetration of the germ tube was also observed. Gem tubes
were observed to have ramified the cut surface of the stem within 9 hours
after inoculation. Substantial enzymatic degradation and hyphae elongation
was also observed 9 hours after inoculation. Cross sections showed that
by 11.2 hours hyphae were observed within the xylem vessels and had penetrated
through the cell walls. However, the lesions indicative of stem blight
were not observed until 24 hours following inoculation.
REVEEW OF SIGNIFICANT LITERATURE
Botrytis blossom blight and leaf spot caused by B. cinerea Pers. ex.
Fr. was first reported on geranium by Melchers in 1918 with complete symptom
description following in 1926 (Melchers). Annual dollar losses in geranium
production nationwide due to B. cinerea are estimated at $5.1 to 7.6 million
based on known figures. Losses in Pennsylvania alone are estimated at $130,000
to $190,000 (personal communication; Oglevee Limited). These estimates
do not include the costs of B. cinerea infection resulting in reduced plant
quality or decreased cutting production. Nichols and Nelson (1982) reported
that Botrytis typically lives on aging tissue such as flowers, leaves,
broken stems and cutting stubs but under appropriate conditions can severely
damage leaves, flowers, and stems of healthy plants, especially if the
tissue is succulent. Leaf lesions on geraniums commonly develop where infected
senescent flower parts have fallen. These lesions enlarge if sufficient
moisture is present becoming irregular in outline and potentially infecting
the entire leaf (Melchers, 1926).
Conidia may be a phylloplane resident resulting in latent infection
under desirable conditions. Conidia are considered the primary source of
infection of cutting stubs and a latent rot infection of cuttings. Nichols
and Nelson (1982) suggested conidia can become lodged on the stem surface
remaining dormant until cuttings are placed in the propagation bench for
rooting. Under these wet and humid conditions, the conidia germinate forming
a cutting rot. Spraying chrysanthemum stock plants with fungicide decreased
subsequent infection on detached cuttings, suggesting stock plants were
an important inoculum source (Smith, 1967).
Moisture and temperature are of primary importance for B. cinerea conidia
germination and subsequent infection, although Good and Zathureczky (1967)
found that conidia were very tolerant of drying. Melchers (1918) suggested
in his original description of B. cinerea on geranium that an abundance
of moisture and insufficient ventilation favored this disease. Similarly,
leaf and flower blight was observed on geranium in glasshouses and in sheltered
places outdoors in southern California, especially during rainy weather
(Baker, 1946). Losses of greenhouse tomatoes and cucumbers were estimated
at 60 to 75% during a season of damp, cloudy weather due to Botrytis stem
and fruit rots and blossom blight which were generally considered of minor
importance (Kadow, et al., 1993).
Lesion growth on geraniums was reported to increase with temperatures
from 10 to 25 C. At 30 C typical Botrytis lesions and sporulation on geraniums
did not occur (Hyre, 1972). DeLozier (1980) showed sporulation to be greatest
at 15 to 20 C with 100% RH. At 100% M temperatures of the range 10 to 30
C had no significant effect on the number of plants infected, the latent
period, or the rate of lesion growth.
Similarly, the rate at which artichoke bracts decay following inoculation
increased with temperatures from 0 to 20 C, although the increase was not
uniform (Lipton and Harvey, 1960). Heavy B. cinerea infection of static
flowers, bracts and scape wings occurred at 24 C with moderate infection
at 20 and 28 C (Jackson, 1960). The optimum temperature for the leaf-spotting
phase of gladiolus was approximately 55 to 65 F with slightly less infection
at 40 to 45 F (McClellan, et al., 1949).
Conversely, poinsettias grown at continuous 10 C showed a significant
increase in incidence of B. cinerea in comparison to plants grown at 17
C continuous temperature (Sammons et al., 1982). Similarly, disease incidence
of macadamia racemes was negatively correlated with temperatures above
22 C (Hunter and Rohrbach, 1969).
OBJECTIVES and POTENTIAL BENEFITS
Data gathered thus far by the principle investigator clearly shows that
modification of the microenvironment can reduce incidence of disease caused
by B. cinerea. Data presented in the background information section of
this proposal show that heated air forced into a dense plant canopy via
perforated PVC pipe placed among stock plants geranium canopy for 6 hours
per day reduced stem blight and sporulation of B. cinerea. Similarly, heated
air forced into a dense plant canopy via a perforated plastic tube under
an open-bottom bench located within a commercial greenhouse 24 hour per
day effectively reduced stem blight and sporulation of B. cinerea. However,
in order to incorporate this technology into current computerized environmental
control units and minimize costs, the minimum level and duration of temperature
and relative humidity necessary to interrupt the disease cycle of B. cinerea
on senescent leaves and wounded stems must be defined. Therefore, the following
objectives have been defined: Years 1 and 2
1. Verify where known and determine where not known the level and duration
of temperature and relative humidity necessary to interrupt the disease
cycle of B. cinerea on (a) necrotic nbsp; geranium leaves and (b) wounded
stems of geranium stock plants.
Information gathered thus far indicates the following. Conidia of B.
cinerea begin to germinate within 60 minutes on a wet leaf surface at the
optimum temperature of 20 C. Within four hours, 40% of the conidia germinate
and by 6 hours 77% of the conidia germinate. This information is critical
because it represents what would occur in a grower situation on plant tissue
and provides the groundwork for a predictive system.
Germination and penetration of conidia on a wet wounded stem occurs
within 3 hours at 20 C. Enzymatic degradation at the point of penetration
of the germ tube occurs. Germ tubes ramify the cut surface of the stem
within 9 hours. Substantial enzymatic degradation and hyphae elongation
also occurs within 9 hours. Within 12 hours, hyphae occur within the xylem
vessels and penetrate through the cell walls. However, the lesions indicative
of stem blight are not observed until 24 hours following ineculation at
which time current control methods appear ineffective.
Years 2 and 3
2. Based upon studies conducted under objective 1, construct computerized
software to regulate the level and duration of temperature and relative
humidity necessary to interrupt the disease cycle of B. cinerea
3. Validate the computer software within a research and commercial greenhouse.
In the 1970’s, energy and labor costs increased dramatically, physical
al facilities aged, and the ability of the industry to absorb an exploding
information base and, hence, to adopt an increasingly complex technology,
declined. This allowed other nations to gain a competitive edge and for
the first time in our history, imports from other countries commanded a
significant portion of our market. As we move toward the 21st century,
a concerted effort must be made to regain the initiative. Many of the issues
are related to market access and marketing, but improving demand will not
meet the challenge if that demand is supplied by lower cost inputs.
Only through the production of a cost-effective, high quality product
delivered in the quantities wanted and at the time they are wanted will
the industry become revitalized. To achieve this goal the following two
objectives must be met: (1) pathogen-free planting stock, and (2) computerized
plant growth optimization strategies. This research proposed pertains to
the achievement of both of these goals.
Increasing the number of stock plants per unit area could increase the
production of cuttings. If spacing of stock plants could be decreased without
an increase in B. cinerea, cutting quality and production could increase
the competitiveness of U.S. growers. A stock plant system is envisioned
whereby heated air can be forced into a dense geranium canopy at critical
intervals for specific durations to interrupt the disease cycle of B. cinerea
on senescent leaves and wounded stems. The application of the forced heated
air could be accomplished via perforated plastic tubes underneath an open-bottom
bench or PVC pipes placed on top of the pots within the plant canopy. The
intervals and duration of the heated air could be regulated by a computerized
environmental monitoring system. It is proposed that this method of growing
stock plants would allow an increased number of stock plants per unit area
and increase the production and quality of cuttings. The technology of
altering the microenvironment among closely-spaced stock plants to interrupt
the disease cycle of B. cinerea could apply not only to geraniums, but
to all growing systems utilizing stock plants for cutting production.
Once the environmental parameters preceding an epidemic are gathered
and formulated as a predictive model, this information could be utilized
in any growing system. In crops where a dense canopy is not formed, it
is probable that altering the RH and or temperature would be adequate for
averting an epidemic when environmental conditions would otherwise be favorable.
MATERIALS AND METHODS Objective 1. Accomplished in years 1 and 2 of
project
Verify where known and determine where not known the level and duration
of temperature and relative humidity necessary to interrupt the disease
cycle of B. cinerea on (a) necrotic geranium leaves and (b) wounded stems
of geranium stock plants.
Dry B. cinerea conidia will be used as inoculum. Conidia will be collected
from 10 to 14-day old sporulating cultures into a pipet under a very low
vacuum and tapped out of the pipet onto weighing paper. A conidia settling
tower will be used to inoculate the plant material.
1. a1) Influence of Temperature on Conidia Germination and Infection
Four plants will be inoculated and placed in the dew chamber for 24
hours at 15, 20, 25 C. One leaf per plant will be chosen on the basis of
age (oldest leaf). Four 1-cm2 leaf pieces will be removed from the leaf
specimen, fixed in formalimacetic acid (FAA) stained and examined. Conidia
with swollen germ tube tips and hyphae within lesions will be counted.
1. a2) Conidia Germination Rate on Leaf Surfaces
Plants will be inoculated and placed in the dew chamber at 15, 20, and
25 C. After 1, 2, 4, 6, 8, 10, 12, and 24 hours, 4 1-cm2 leaf pieces from
one previously selected leaf of each of 4 plants will be removed, fixed
in FAA, stained, and germinated conidia counted.
1. a3) Longevity of Conidia on Leaves
Plants will be inoculated and placed in a growth chamber at 15, 20,
and 25 C with 60 + 10% RH and a 16 hour photoperiod. After 0, 1, 2, 4,
or 8 days, 6 plants will be removed from the growth chamber and incubated
for 36 hours in a dew chamber at 15, 20, 25 C with a 12 hour photoperiod.
The number of lesions per plant will be counted and percent leaf area infected
will be determined with a leaf area meter 24 hours after removal from the
dew chamber.
1. a4) Influence of Dew Period on Hyphal Development
Inoculated plants will be placed in the dew chamber for 7, 4, and 6
days of continuous dew at 15, 20, 25 C with a 12 hour photoperiod. Randomly
identified leaf sections containing lesions will be removed from the plants,
fixed in FAA, cleared in 70%o ethanol and stained in 1% aqueous trypan
blue. Lengths of hyphae will be measured with an ocular micrometer.
1. a5) Influence of Dew Period and Temperature on Lesion Production
A group of 24 plants will be inoculated and placed in a dew chamber
at temperatures of 15, 20, and 25 C. Six randomly identified plants will
be removed after exposure to 5, 10, 20, and 40 hours of continuous dew
and moved to a growth chamber set at the same temperature as the dew chamber
treatment with 60 + 10% RH and 16 hour photoperiod. Lesions will be counted
on each plant after variable times in the dew chamber and growth chamber
total 72 hours. Percent leaf area infected will be determined with a leaf
area meter.
1. a6) Influence of Timing of Dew Period Interruption on Lesion Production
Plants will be inoculated and incubated in a dew chamber at 15, 20,
or 25 C for 2, 41 13@ 16@ and 24 hours. Incubation periods are to be followed
by 2 hours without dew in a 15, 20, or 25 C growth chamber at 50 + 10%
RH and continuous light. Following the interruption period, plants will
be moved back to the dew chamber for the remainder of the 24 hour incubation
period. Inoculated and noninoculated controls will remain in the dew chamber
for 24 hours. The number of lesions will be counted after an additional
24 hours in the growth chamber. During the 24 hours in the growth chamber,
a 16 hour photoperiod will be implemented. Percent leaf area infected will
be determined with a leaf area meter. 1. a7) Influence of Interrupted Dew
Periods on Lesion Production
Inoculated plants will be incubated without light in a dew chamber for
6 hours and then transferred to a growth chamber at 15, 20, or 25 C, RH
of 65 + 10% and continuous light After 30, 60, 90, 120, and 150 minutes
in the growth chamber, 6 replicate plants for each interruption period
will be returned to the dew chamber for the remainder of the 24 hour incubation
period. Noninoculated controls will remain in the dew chamber for 24 hours.
Lesions will be counted after an additional 24 hours in the dew chamber.
Percent leaf area infected will be determined with a leaf area meter. During
the 24 hours in the dew chamber, a 12 hour photopexiod will be implemented.
The experiment will be repeated by interrupting the dew chamber environment
for 4, 8, 12, and 24 hours after which the plants will be returned to the
dew chamber for an additional 48 hour incubation period. During the 48
hours in the dew chamber, a 12 hour photoperiod will be implemented.
1. a8) Influence of Humidity During an Interruption
Inoculated plants will be placed in the dew chamber for 6 hours. Six
replicate plants will be removed and placed in the growth chamber at 30,
60, and 90% RH and continuous light for 20 minutes and returned to the
dew chamber for the remainder of the 24 hour period. Control plants will
remain in the dew chamber for the remainder of the 24 hour period. Control
plants will remain in the dew chamber continuously for 24 hours. Lesions
will be conducted after an additional 24 hours in the growth chamber. During
the 24 hours in the growth chamber, a 16 hour photoperiod will be implemented.
Percent leaf area infected will be determined with a leaf area meter.
1. b1-8) Studies Investigating Infection and Progression of B. cinerea
on Wounded Stems of stock Plants.
Experiments described above utilizing leaf tissue will also be conducted
with wounded stems. Disease progress will be monitored by measuring visual
darkened lesions on the stem. Microscopic observations will be conducted
using the scanning electron microscope. Cuttings will be excised from stock
plants 2 cm above a plant node using flame sterilized razor blades. Dry
B. cinerea conidia will be used as inoculum. Transverse cuts will be made
at a plane 5 mm from the inoculated surface with flame-sterilized blade.
Samples will be fixed in 3% glutaraldehyde in 0.15M sodium cacodylate (pH
7.1) and refrigerated overnight. All samples will be postfixed in osmium
tetroxide vapors for 1 hr, followed by a graded ethanol dehydration series
and storage in 100% ethanol at room temperature for 15 hours prior to drying.
Stem samples to be observed along the longitudinal axis will be cryofractured
and subsequently thawed in 100% ethanol. All samples will be critically
point dried using bone dry liquid carbon dioxide. Dried samples will be
mounted onto 13 mm aluminum stubs with adhesive tabs and silver dag, then
sputter coated with 42 nm of gold. The prepared stubs will be examined
with a scanning electron microscope and representative photomicrographs
taken.
Objectives 2 and 3. To be accomplished during years 2 and 3 of project
Based upon studies conducted under objective 1 and data collected during
1984-1987 from a commercial greenhouse, construct computerized software
to regulate the level and duration of temperature and relative humidity
necessary to interrupt the disease cycle of B. cinerea. Validate the computer
software within a research and commercial greenhouse.
The interpretation of information gathered in objective 1 will be used
to develop algorithims. In addition, environmental parameters and corresponding
atmospheric concentrations of conidia collected during 1984-1987 in a commercial
greenhouse will be analyzed. Dr. Mel Lacy of the department of Botany and
Plant Pathology at Michigan State University has recently formulated a
program to time fungicide sprays for control of Botrytis squamosa that
causes leaf blight of onion using a conidial release predictor (Lacy, 1991).
Due to the high rate of success and acceptance of this program among onion
growers, a similar strategy will be employed for Botrytis cinerea on geraniums
and other ornamental crops. This predictive system is envisioned to be
based on a table with sporulation index values and will be created from
data collected from 1984-1987 and from studies conducted in years 1 and
2 of this proposal. Average temperature and average vapor pressure deficit
during the previous 72 hours will be used to establish a threshold to trigger
a spray or modify the environment. The precision of the algorithims will
be evaluated within a research and commercial greenhouse.
FACILITIES AND EQUIPMENT AVAILABLE
Much of the equipment necessary for this research project is located
in the Investigator’s laboratory or in the Department of Botany and Plant
Pathology located in the Plant Biology Building.
Laboratory equipment includes laminar flow hoods, autoclave, distilled
water system, clinical centrifuge, pH meter, incubated orbital shaker,
2 incubators, phase contrast and inverted phase contrast microscopes, analytical
balances and miscellaneous instruments and supplies. The facilities include
a greenhouse (14,000 sq. ft) containing 12 benches and is equipped with
supplementary lighting.
LITERATURE CITED
Baker, K.F. 1946. Observations on some Botrytis diseases in California.
Plant Dis. Rep. 30:145- 155. Cline, M.N, and Neely, D. 1983. Wound-healing
process in geranium in relationship to basal stem rot caused by pythium
ultimum.
DeLozier, K.M. 1980. The effect of ambient temperature and relative
humidity on Botrytis cinerea Pers. on Pelargonium x hortorum Bailey. M.S.
Thesis, Penn. State
Dennis, C., and Davis, R.P. 1979. Tolerance of Botrytis cinerea to iprodione
and vinclozolin. Plant Pathol. 28:131-133. Good, H.M., and Zathureczky,
P.G.M. 1967. Effects of drying on the viability of germinated spores of
Botrytis cinerea. Phytopathology 57.719-722.
Heursel, J, and Kamoen, O. 1976. Technical aspects of the storage of
azalea and rhododendron cuttings in a cold store. Meded. Rijksstation Sierplantenteelt.
37:1-8.
Hunter, J.E., Rohrback, KG., and Kunimoto, R.& 1972. Epidemiology
of Botrytis blight of macadamia racemes. Phytopathology 62:316-319.
Hyre, R.A. 1972. Effect of temperature and light on colonization and
sporulation of the Botrytis pathogen on geranium. Plant Dis. Rep. 56:126-130.
Jackson, C.R. 1960. Crown rots and Botrytis flower blight of statice.
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BUDGET
The following budget is proposed to fund the conclusion of the first
phase and the completion of the second phase of this proposal:
Fungal Culture Media and Petri Plates
$ 600
Greenhouse Supplies:
Fertilizer, Soilless Media, Pots,
$ 900
Heaters, Fans, Plastic Tubing
$ 700
Computer Programmer
$ 7,020
($9.00/hr x 15 hr/wk)
Computer Supplies
$ 50
Disks
Graduate Student Stipend/Technical Support
$15,250
($12,500 + 22% fringe benefits)
Scanning Electron Microscopy Usage
$ 2,800
($10.00/hr beam time)
Scanning Electron Microscopy Supplies
$ 156
(Film $7.79/8 exp.)
Growth and Dew Chamber Usage and Supplies
$ 1,800
($50/unit/month x 3 units)
——————————————————————————–
TOTAL
$29,276
PROJECT LEADER QUALIFICATIONS
Dr. Mary Hausbeck has recently graduated from The Pennsylvania State
University (PSU) under the direction of Dr. S.P. Pennypacker, a noted epidemiologist.
She earned a B.S. in Horticulture and completed a M.S. in Horticulture/Plant
Pathology under the direction of Drs. R.D. Heins and C.T. Stephens at The
Michigan State University (MSU). Dr. Hausbeck completed a 6 month post-doctoral
project involving the tomato spotted wilt virus (TSWV) on ornamentals at
PSU prior to her appointment as a Visiting Assistant Professor at MSU.
Her current responsibilities at MSU include greenhouse crops.
She is the senior author on 5 manuscripts published in refereed journals
concerning crown and root rot of geraniums caused by Pythium ultimum documenting
etiology, symptomatology, fungicide efficacy, and cultivar resistance.
She is also the senior author on 3 manuscripts accepted and 3 manuscripts
in review, all of which address various aspects of Botrytis cinerea. A
Disease Note concerning TSWV is also published and a manuscript concerning
TSWV is currently in review. There are 9 published abstracts concerning
her work on greenhouse crops.
SUMMARY
Controlling disease caused by B. cinerea through modification of the
greenhouse environment is an attractive addition to traditional control
methods. Although event-driven, interactive computers are available with
the capacity for predicting disease epidemics and altering the greenhouse
environment, the data base necessary for the development of software that
will provide predictions with a high confidence level is lacking.
Data gathered thus far by the principle investigator clearly shows that
modification of the microenvironment can reduce incidence of disease caused
by B. cinerea. A stock plant system is envisioned whereby heated air can
be forced into a dense geranium canopy at critical intervals for specific
durations to interrupt the disease cycle of B. cinerea on senescent leaves
and wounded stems. The application of the forced heated air could be accomplished
via ; perforated plastic tubes underneath an open-bottom bench or PVC pipes
placed on top of the pots within the plant canopy. The intervals and duration
of the heated air could be regulated by a computerized environmental monitoring
system. It is proposed that this method of growing stock plants would allow
an increased number of stock plants per unit area and increase the production
and quality of cuttings. The technology of altering the microenvironment
among closely-spaced stock plants to interrupt the disease cycle of B.
cinerea could apply not only to geraniums, but to all growing systems utilizing
stock plants for cutting production. Once the environmental parameters
preceding an epidemic are gathered and formulated as a predictive model,
this information could be utilized in any growing system. In crops where
a dense canopy is not formed, it is probable that altering the RH and or
temperature would be adequate for averting an epidemic when environmental
conditions would otherwise be favorable.
However, in order to incorporate this technology into current computerized
environmental control units and minimize costs, the minimum level and duration
of temperature and relative humidity necessary to interrupt the disease
cycle of B. cinerea on senescent leaves and wounded stems must be defined.
Years 1 and 2
1. Verify where known and deterinme where not known the level and duration
of temperature and relative humidity necessary to interrupt the disease
cycle of B. cinerea. on (a) necrotic geranium leaves and (b) wounded stems
of geranium stock plants.
Years 2 and 3
2. Based upon studies conducted under objective 4 construct computerized
software to regulate the level and duration of temperature and relative
humidity necessary to interrupt the disease cycle of B. cinerea
3. Validate the computer software within a research and commercial greenhouse.
