Analysis of Factors
Affecting Latent Infection and Sporulation of Monilinia fructicola on Prune fruit
Yong Luo, Zhonghua Ma, and Themis
J. Michailides, Department of Plant Pathology, University of California,
Davis, Kearney Agricultural Center, Parlier, CA 93648
Corresponding
authors: Yong Luo and Themis J. Michailides
E-mail:
yluo@uckac.edu and themis@uckac.edu
ABSTRACT
Luo,
Y., Ma, Z., and Michailides, T. J. 2001. Analysis of factors affecting latent
infection and sporulation of Monilinia
fructicola on prune fruit. Plant Dis. 85:xxx-xxx.
Two studies were conducted to determine
the effects of water content (WC) on sporulation on thinned fruit and the
effects of wetness duration, inoculum density, and temperature on secondary
infection of prune fruit by Monilinia
fructicola, the main causal pathogen of brown rot in California. In the
first study, sporulation intensity and duration of sporulation of the pathogen
were tested on inoculated thinned fruit with five levels (67.2, 53.8, 40.3,
26.9 and 13.4%) of WC. Regression analyses showed that both sporulation
intensity and duration of sporulation increased as WC of thinned fruit
increased. The predicted difference in duration of sporulation between fruit
with 13.4 and 67.2% WC was about 3 days. In the second study, three inoculum
concentrations (8,000, 16,000, and 24,000 conidia/ml) of M. fructicola were atomized onto prune fruit on trees in an
orchard. Inoculated fruit and shoots were covered with plastic bags to maintain
wetness duration for 4, 8, 12, or 16 h. An overnight freezing and incubation
technique was used after harvest to determine the proportion of fruit with
latent infection. Regression analysis demonstrated that inoculum concentration
and wetness duration were significant factors affecting secondary infection.
Temperature was less important. Increased inoculum concentration and wetness duration
increased the percentage of fruit with latent infections. Increased temperature
decreased the percentage of fruit with latent infections.
Additional
keywords: epidemiology, Prunus domestica,
quiescent infection, stone fruit
Brown rot, caused by Monilinia fructicola (G. Wint.) Honey, is a destructive disease of
stone fruit (Prunus spp.) (1,3,14,22). Ascospores
or conidia produced from mummies infected by M. fructicola serve as inoculum sources that cause blossom blight
in the spring (3,14,21). These primary infections can provide inoculum for
latent infection of fruit (6,12,23). Non-abscised, aborted fruit in trees and thinned
fruit on the orchard floor are important sources of secondary inoculum for fruit
brown rot (1,14,18). The significance of thinned fruit as a source of
secondary inoculum in California nectarine orchards was also confirmed (9). Infected thinned fruit on the floor served as a
source of spores that cause secondary infection, and sporulation of M. fructicola on these fruit was
significantly correlated with the severity of preharvest and postharvest brown
rot (8,9). Cultural practices in orchards, especially
irrigation and management of thinned fruit, may affect water content (WC) and
decomposition of thinned fruit. Estimation of inoculum potential from thinned
fruit could be used to guide disease control. Information on how sporulation
relates to WC of thinned fruit is needed to devise control strategies and
reduce secondary infection. Appropriate cultural practices in orchards that
affect thinned fruit and their WC might be implemented to reduce secondary
inoculum.
Under favorable conditions, conidia of M. fructicola are dispersed in the air (13), deposited on the fruit surface, and cause infection
(2,4,5,15,19). Inoculum
concentration and environment serve as important factors affecting secondary
infection. Effects of temperature and wetness duration on infection of fresh or
harvested stone fruits have been reported (2,4,19). Biggs and Northover (2) found that the optimum temperatures and wetness
durations were 20 - 22.5ºC and 18 h for infection of cherry fruit, and 22.5 -
25ºC and 12 h for infection of peach fruit. Inoculum concentration
significantly affected brown rot development on detached cherry (18), nectarine, and peach fruits (11). However, little information is available about
effects of these factors on brown rot development of fruit on trees. Since the physiological conditions between
detached fruit and fruit on trees are different, how fruit infection on trees
relates to different environments is important for estimating inoculum
potential and predicting disease development in the field.
If the inoculum potential from thinned
fruit could be determined based on the WC of these fruit, then the risk of
secondary infection could be more precisely predicted using the relationship
between inoculum availability and predicted temperature and wetness duration.
The objectives of this study were 1) to determine the effect of WC of thinned,
infected prune fruit on sporulation of M.
fructicola, and 2) to define the effects of inoculum concentration, wetness
duration, and temperature on secondary infection of prune fruit.
MATERIALS AND METHODS
Effects of WC on sporulation of M. fructicola on thinned fruit.
An isolate of M. fructicola collected from a prune orchard at the Kearney
Agricultural Center, University of California in Parlier, was used in this
study. This isolate was cultured on potato dextrose agar (PDA) amended with a
25% v/v lactic acid (2.6 ml/L). Cultures were incubated at 23 ± 2ºC for 5 days
in the dark. Conidia of M. fructicola
were harvested by pouring 3 ml sterile distilled water in each Petri dish, and
the spore concentration was adjusted to 1,000 conidia/ml using a hemacytometer.
Immature
fruit were collected from a prune orchard at Kearney Agricultural Center. The
experiments were conducted from July to August 1999, and fruit stages were from
late embryo growth to first harvest (20). Fifteen green prune fruit (cv. French) of different
sizes (13.5 ± 3.4 g, 39.2 ± 4.4 mm length and 27.1 ± 1.9 mm diameter) were
arbitrarily selected to determine the dry weight of fruit. Each fruit was
weighed fresh and then dried at 65ºC for about 3 days to stabilize the weight.
The average percentage of dry fruit weight over the fresh fruit weight from the
15 fruit was 32.8 ± 1.1 %. This value was used to calculate the water content
of fruit for subsequent experiments.
Fresh prune fruit collected from the same orchard were
surface-disinfested with 0.525% sodium hypochlorite (10% commercial bleach) for
3 min and rinsed with sterile distilled water five times. To initiate latent
infections, a sterile nail was used to make a wound (3 mm diameter and 2 mm
depth) on the surface of each fruit, and a 30-μl drop of spore suspension
(1,000 conidia/ml) was placed on each wound. The inoculated fruit were placed
on waxed wire screens in sterile plastic containers (40 by 24 by 12 cm) and 200
ml water was added to the container to increase humidity and facilitate
infection. Containers with the fruit were incubated at 23ºC for 2 days to
establish latent infection, then fruit were removed from the containers and
placed into an open wooden frame box (44 by 35.5 cm) on top of paper towels to
dry under ambient outdoor conditions (about 6 to 8 h/day under direct
sunlight). No further development of brown rot was apparent on the fruit. Five
of these fruit were arbitrarily selected and individually weighed daily to
determine WC loss. The percentage WC of fresh fruit was 67.2%. When the average
WC of the selected five fruit reached 53.8%, 40.3%, 26.9 and 13.4%
(corresponding to 80, 60, 40, and 20% of the WC of fresh fruit, respectively),
35 fruit with each corresponding WC were collected and surface-disinfested with
0.525% sodium hypochlorite for 5 min, rinsed with sterile distilled water five
times, placed on waxed wire screens in sterile plastic containers, and
incubated at 23ºC and 98% relative humidity for the following experiments.
Inoculated fresh fruit (without drying) were used to represent the fruit with
67.2% WC.
The
day when at least 50% of the 35 fruit showed sporulation was used as starting
date of sporulation for each WC/drying time treatment. At that time, five fruit
covered with spores were chosen for testing sporulation intensity. All spores
were washed from the five fruit with sterile distilled water 1 day prior to
quantifying sporulation. After washing, fruit were incubated for one more day,
and the new spores formed were washed into 200 ml distilled water using a
paintbrush. The resulting spore suspension was shaken at 180 osc/min for 10 min
to break chains of spores and the number of spores of M. fructicola for each of the five fruit was determined using a
hemacytometer. Sporulation measurements on the same five fruit were repeated 2
and 5 days after sporulation began.
From the remaining 30 fruit, those
showing at least 25% surface area covered with sporodochia producing spores
were recorded daily as fruit with sporulation. Following daily observation,
spores were brushed off from the fruit and the fruit were returned to the
plastic container and incubated until no new conidiophores were visually
observed. Each experiment lasted about 10 to 12 days. The fruits used in each
experiment were at the same growth stage. The experiment was conducted four
times.
Treatments were arranged in a
randomized complete block design (7). Average sporulation intensity per fruit per day was
calculated from the two measurements for each tested fruit. Analysis of
variance was conducted to determine the significance of variance in sporulation
from experiments as blocks and from WC of thinned fruit as the treatments. The
interaction between these two factors was used as an error. The ANOVA procedure
of SAS (Version 7.0, SAS Institute, Cary, NC) was used in this analysis.
Average sporulation intensity calculated from five sub-samples for each
experiment was used in regression of sporulation on WC by using the computer
software SigmaPlot (version 5.0, SPSS inc., Richmond, CA).
In each experiment, the duration of
sporulation for each WC was determined as days from the sporulation starting
date through the day when 90% of fruit stopped producing spores. This duration
of sporulation was determined for each WC in each experiment and was linearly
regressed on WC of thinned fruit using the REG procedure of SAS. To describe
the dynamic process of sporulation, the proportion of fruit with sporulation of
M. fructicola over the corresponding days after incubation was used to
obtain a linear regression equation for each WC using the REG procedure of SAS.
Homogeneity of regression coefficients between any two regressions was tested (7).
Effects of wetness duration, inoculum concentration, and
temperature on latent infection of prune fruit.
Inoculum was prepared as described previously. Spore concentrations used
in this study were adjusted to 8,000, 16,000, and 24,000 conidia/ml. A total of
500 ml spore suspension of each of the three concentrations was made.
A split-plot design was used in this
study. Inoculations were conducted in a prune (cv. French) orchard at the
Kearney Agricultural Center in July when fruit were between late embryo growth
and first harvest (20). The fruit size was similar to those of the above
study. Branches of prune trees with 20 to 30 fruit were uniformly sprayed with
30 ml spore suspension of each concentration using a hand-held sprayer (ACE
Hardware Corporation, Oak Brook, IL). Immediately after inoculation, each
branch was closed tightly in a 1.5 mil transparent plastic bag to maintain high
relative humidity around the inoculated branch. The three inoculum
concentrations were applied to different branches of the same tree with enough
distance between them to protect from cross contamination. A non-inoculated
control was established by spraying 30 ml sterile distilled water on a branch
of a separate tree.
For each experiment, inoculation was
conducted at about 20:00 PST. Four treatments consisting of wetness durations
of 4, 8, 12, and 16 h were accomplished by uncovering the plastic bags at 0:00,
4:00, 8:00 and 12:00 PST, respectively, on each of four inoculated trees. Fruit
normally dried within 15 to 20 minutes after bags were removed. Three branches
served as sub-samples for each inoculum concentration and each wetness
duration, and one sample of water-inoculated control for each wetness duration
was used in each experiment. To prevent high temperature in the inoculated bags
caused by sunlight, the plastic bags for treatments of 16-h wetness duration
were covered with paper bags (30.4 by 17.3 by 36.2 cm) from 8:00 PST until completion
of the wetness duration. A data logger (ONSET Company, Pocasset, MA) was used
to record hourly temperature outside but close to the bags during each
experiment. However, the temperatures measured were probably a few degrees
lower than those inside the bags. At the end of each wetness duration, the
plastic bags were removed and the inoculated branches were marked. No natural
dew was observed during the experiments. Five experiments were conducted using
similar methodology. Hourly temperatures during the wetness duration for each
inoculation are shown in Fig 1.
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At commercial harvest time in August,
inoculated fruit showing no disease were harvested separately for each
treatment combination of inoculum density and wetness duration. The overnight
freezing incubation technique (16,17) was used to determine the proportion of fruit with
latent infections. Fruit were
surface-sterilized in a chlorine solution (32 ml of 0.525% sodium hypochlorite,
32 ml 95% ETOH, and 0.01ml surfactant Tween-20 in 2 liters water) for 5 min.
The fruit were then washed with sterile distilled water five times and placed
on a waxed wire screen in a plastic container (40 by 24 by 12 cm) with water at
the bottom to increase relative humidity. The containers were placed in a
freezer at -16ºC for 10 h and then transferred to a laboratory bench at 23 ± 2ºC for 7 days. The proportion of fruit
covered with sporulation of M. fructicola
was recorded.
The proportion of fruit with latent
infection was transformed with the arc sine transformation prior to statistical
analysis (7). Analysis of variance was conducted using the ANOVA
procedure of SAS with the experiment treated as replication, inoculum
concentration treated as the main-plot factor and wetness duration treated as
the subplot factor (7). Average temperature for each wetness duration was
calculated for each experiment. In order to study the effects of inoculum
concentration on latent infection, experiments were combined into one data set
for each inoculum concentration. Linear regression was conducted using the REG
procedure of SAS for each inoculum concentration. The proportion of fruit with
latent infection was treated as the dependent variable and the wetness duration
and temperature as independent variables.
All combined data were also used for an overall regression analysis with
the proportion of fruit with latent infection as the dependent variable and
inoculum concentration, wetness duration, and temperature as independent
variables.
RESULTS
Effects of WC on sporulation of thinned
fruit. Water content (WC) had a significant
effect on sporulation (P = 0.0002)
while the experiment (block) did not. An equation describing the amount of
sporulation in relation to the WC was obtained (Fig. 2A).
A significant linear relationship between WC and duration of sporulation
in days (DS) was also obtained (Fig. 2B). Duration of sporulation was prolonged
with increased WC. Sporulation on fruit with 67.2% WC (fresh fruit) lasted 7 to
9 days (with an average of 8 days). The predicted difference in average
duration of sporulation between fruit with 67.2% and 13.4% WC was about 3 days.
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On the first day after incubation, 100% of fruit with 67.2% WC produced
spores (Fig. 3), but this proportion varied on fruit with 53.8% WC and 40.3% WC
(Fig. 3), and was decreased to less than 85% on fruit with 26.9% WC (Fig. 3).
About 5 days after incubation, 70, 60, 60, and 50% fruit, with 67.2, 53.8,
40.3, and 26.9% WC, respectively, were predicted to show sporulation of M.
fructicola (Fig. 3). Generally, sporulation of fruit with higher WC was
greater than that of fruit with lower WC during the first 5 to 6 days of
sporulation. For example, only 20% fruit with 13.4% WC produced spores after 6
days of incubation (data not shown).
Linear relationships between days after incubation of fruit (D) and
percentage of fruit with sporulation (%) (SP) were obtained for the four levels
of WC (Fig. 3). Analysis of homogeneity of regression coefficients demonstrated
that the absolute value of the regression coefficient (slope) for 67.2% WC
(Fig. 3) was significantly greater (P <
0.05) than those of regressions for 53.8% WC and 26.9% WC (Fig. 3). The
absolute value of the regression coefficient for 26.9% WC was significantly
smaller (P < 0.05) than those of
the other three regressions. Regression coefficients for 40.3 % WC and 53.8% WC
were not significantly different (Fig. 3).
 
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Effects of
wetness duration, inoculum concentration, and temperature on latent infection
of prune fruit. None of the
non-inoculated control fruit developed latent infection. Analysis of combined
data from all experiments showed significant effects for experiment (block) at P < 0.0001, wetness duration at P = 0.0196, and inoculum concentration
at P < 0.0001. The interaction between wetness duration and
inoculum concentration was not significant, and errors of wetness duration and
inoculum concentration were also not significant.
Regression analysis demonstrated that
when inoculum concentration of 8,000 conidia/ml was used, wetness duration had
a significant impact on fruit with latent infections (P = 0.008), but temperature did not (P = 0.86). Similar results were obtained when 16,000 conidia/ml was
used, that wetness duration had significant effects on latent infection (P = 0.0008) while temperature did not (P = 0.23). Both regressions were
significant at P < 0.05. However,
when inoculum concentration was increased to 24,000 conidia/ml, wetness
duration was not significant (P =
0.23) while temperature became significant (P
= 0.037). The overall regression from the combination of inoculum densities
showed that wetness duration and inoculum concentration were both significant
at P < 0.0001 and temperature was
significant at P = 0.043. The
regression was significant at P <
0.0001 with R2 = 0.2624. The regression equation was PLI =14.4 +
1.11IC - 0.78T + 1.13 WD, where PLI is proportion of latent infection (%), IC
is inoculum concentration (103 conidia/ml), T is temperature (ºC),
and WD is wetness duration (h).
DISCUSSION
Results of the first experiment
indicated that increasing WC of thinned fruit led to a greater sporulation
intensity and longer duration of sporulation. Increased inoculum production
from fruit with high WC may increase potential for secondary infections of
prunes. Results of the second experiment indicated that inoculum concentration,
wetness duration, and temperature affect secondary infection of prune fruit.
Increasing inoculum concentration, prolonging wetness duration, and decreasing
temperature within the range of 15 to 30ºC all resulted in more latent
infections.
The relationships between WC of thinned
fruit and sporulation obtained in this study may be used to estimate how much
and how long sporulation will be reduced on fruit with less than 67.2% WC. However, the results could be used only for
fruit with WC greater than 13.4%. When WC decreased below 13.4%, very few
thinned fruit produced spores. Hong and Michailides (10) observed that mycelial growth and sporulation of M. fructicola were reduced along with
decreasing osmotic potential. This implies that sporulation is significantly
related to relative humidity, which may be affected by water content of fruit.
In this study, we tested relationships
between WC of thinned fruit and sporulation under simulated natural conditions.
Fruit infected by M. fructicola were placed in an outdoor-environment
and sampled periodically at different time periods during the drying process.
Undoubtedly, sporulation of thinned fruit could vary as the period of drying
varies. For example, we found that the period needed to obtain different levels
of WC was significantly correlated with WC (r = -0.89), sporulation (r =
-0.85), and duration of sporulation (r = -0.70).
Differences in WC among thinned fruit may
result from many factors including time, ambient temperature, and relative
humidity during the drying period. This study demonstrated that the WC of
thinned fruit could be predicted based on drying period, temperature, or
moisture. For example, after a rainy
period or following irrigation, fruit may dry more slowly (higher WC) compared
to those after a period of drought and sunlight because of changes in ambient
temperature and moisture. The period needed for thinned fruit to reach a
certain WC might be different under different weather conditions. The ambient temperatures
varied among experiments, implying that the time required to reach each level
of WC also varied. However, the interaction between sporulation of M.
fructicola on fruit and experiment was not significant based on an analysis
of individual fruit data rather than means. Therefore, the information obtained
from this study is useful to predict inoculum potential in orchards based on WC
of thinned fruit that relates to environment and cultural practices.
Prune fruit with higher WC produced
spores over a longer period than fruit with lower WC. Therefore, infected fruit
with higher WC could contribute to a higher risk of secondary infection than
fruit with lower WC. Hong et al. (9) showed that thinned nectarine fruit can serve as an
inoculum source for secondary infection. Controlling WC of thinned fruit could
be used for disease management. Cultural practices in orchards that promote
quick drying of thinned fruit may be helpful to reduce these inoculum sources
for secondary infection. If WC of thinned fruit could be quickly reduced to
lower than 26.9%, fruit would produce few conidia, thus reducing the risk of
secondary infection. Humidity could affect the drying process of thinned fruit
in the fields. Hong et al. (10) found that M.
fructicola sporulated more frequently on thinned fruit in irrigation
trenches than on fruit on the dry berms in tree rows. Thus, fruit thinning
should be scheduled between irrigations, thereby providing enough time for
thinned fruit to dry quickly.
Under field conditions,
inoculum concentration and wetness duration were critical factors that
determined the levels of secondary infections of prune fruit. High inoculum
concentration combined with long wetness duration led to more fruit with latent
infections than did lower inoculum concentration with shorter wetness
duration. When inoculum concentration
is low, wetness duration could be a critical factor in severity of infection.
The relationship between spore concentration in the air and on the fruit is
needed to quantitatively estimate the
inoculum potential in the fields.
Temperature was shown to be an
important factor at the highest inoculum concentration tested (e.g., 24,000 conidia/ml). Since these
field experiments were conducted overnight when temperatures were within the
range of those required for M. fructicola
to cause infection (3, 19), the conclusions of this study are applicable only
under temperature conditions similar to those in California. Furthermore, the
temperatures inside the plastic bags were probably a few degrees higher than
those recorded during the incubation periods. More intensive studies on the
effects of temperature on secondary infection are needed for regions where
summer temperatures and levels of moisture are very different from those in
California.
Differences in fruit susceptibility and
temperatures between experiments may have contributed to variability.
Susceptibility of fruit to infection may have changed during the field
experiments. Our recent studies (data not published) showed that fruit
susceptibility to infection increased with fruit growth after the pit hardening
stage. The hourly temperatures among the third, fourth, and fifth inoculations
were relatively consistent, but the temperatures of the first and second
inoculations were higher than those of the other three inoculations (Fig. 1).
Inoculum concentration, wetness
duration, and temperature in the orchard could be used to predict secondary
infection. Biggs and Northover (2) found that on harvested peach fruit, the optimum
temperature for infection was 22.5 to 25ºC and over 70% of the fruit were infected after
12 h of wetness duration at temperatures below 27.5ºC. Corbin (4) reported that on harvested peach, plum, and cherry
fruits, higher inoculum dosage led to shorter incubation periods; and green
fruits needed high inoculum dosages of M.
fructicola for infection of intact fruit, and low inoculum dosages when
fruits were injured. Based on the results of this study, it may be possible to
predict the risk of latent infection based on estimation of sporulation on
thinned fruit and microclimate conditions, such as wetness duration and
temperature.
LITERATURE CITED
1.
Biggs, A. R., and
Northover, J. 1985. Inoculum sources for Monilinia
fructicola in Ontario peach orchards. Can. J. Plant Pathol. 7:302-307.
12.
Jerome, S. M. R. 1958. Brown rot of stone fruits: Latent contamination
in relation to spread of the disease. J. Aust. Inst. Agric. Sci. 24:132-140.