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2006 Plant Management Network.
Accepted for publication 28 September 2006. Published 13 December 2006.


Penicillium expansum Invades Apples Through Stems during Controlled Atmosphere Storage


David A. Rosenberger, Catherine A. Engle, and Frederick W. Meyer, Department of Plant Pathology, New York State (Geneva) Agricultural Experiment Station, Cornell Universitys Hudson Valley Laboratory, P.O. Box 727, Highland, NY 12528; and Christopher B. Watkins, Department of Horticulture, Cornell University, Ithaca, NY 14853


Corresponding author: David A. Rosenberger. dar22@cornell.edu


Rosenberger, D. A., Engle, C. A., Meyer, F. W., and Watkins, C. B. 2006. Penicillium expansum invades apples through stems during controlled atmosphere storage. Online. Plant Health Progress doi:10.1094/PHP-2006-1213-01-RS.


Abstract

Empire apples were collected from six orchards in 1997 and 1998 and were then subjected to various inoculation and storage regimes to determine how non-wounded fruit become infected with Penicillium expansum and to determine if decay susceptibility varies with orchard source. Replicated samples of fruit were inoculated within 24 h of harvest either by placing a 10 l droplet containing 500 conidia of P. expansum onto the end of the apple stems or by placing 500 l containing 10,000 conidia into the stem basin. Fruit were stored for 7 to 9 months either in cold air (1.1C) or in controlled-atmosphere (CA) storage (1.1C, 1.6% oxygen and 2.2% carbon dioxide), and were then evaluated for decay. Twenty-seven to 47% of fruit that had been inoculated by placing spores on the ends of stems developed decay during CA storage whereas less than 1% of similarly inoculated fruit decayed during cold-air storage. Placing spore suspension into the stem basins also resulted in less than 1% decay. Orchard-to-orchard variation in incidence of decay that developed was positively correlated with boron concentrations in both apple leaves (R2 = 0.66) and apple fruit (R2 = 0.62). This is the first report of P. expansum causing commercial losses in apples due to invasion through stems and also the first report suggesting a relationship between boron nutrition and decay susceptibility in apples.


Introduction

Penicillium expansum Link ex. Thom, the cause of blue mold, is the most important postharvest pathogen of apples (16). Postharvest losses in apple storages in the United States were estimated at more than $4.4 million in 1992 (17). Since the early 1970s, P. expansum has been controlled through postharvest applications of thiabendazole (TBZ), benomyl, or thiophanate-methyl (2,7,22). Strains of P. expansum developed resistance to the benzimidazole fungicides soon after the fungicides were introduced (1,19,29,32). However, postharvest treatments remained effective because benzimidazole fungicides were applied in combination with the antioxidant chemical diphenylamine (DPA) which was used to prevent superficial scald (25). DPA suppressed strains of P. expansum that were resistant to benzimidazole fungicides (20). As a result, the benzimidazole/DPA combination remained effective long after benzimidazole fungicides were no longer effective for controlling field diseases such as apple scab and brown rot of stone fruits (11,12,28).

During the 1990s, losses to postharvest decays gradually increased in apple storages in New York State (17). Two percent of isolates collected from apple storages in the late 1980s were resistant to the benzimidazole/DPA combination (23). However, the prevalence of these double-resistant isolates appears to have increased subsequently as evidenced by disease-control failures that became common in commercial storages where postharvest treatments of TBZ plus DPA were being applied. Losses were especially severe in Empire fruit that were held in controlled-atmosphere (CA) storage, sometimes exceeding 15% in fruit packed after April (17). Even when the incidence of decay was high; however, non-decayed fruit in the same bins were usually still firm and show no signs of internal breakdown or other disorders. Thus, the decay problem could not be attributed to over-maturity or senescent breakdown in fruit from affected lots.

Most of the published literature on blue mold indicates that P. expansum is primarily a wound pathogen (16). However, Empire fruit that developed blue mold decay during CA storage in New York often had no wounds or skin abrasions that could have provided an entrance site for P. expansum. The manager of a large apple storage in Albion, NY noted that fruit from some orchards consistently developed more decay than fruit from other orchards stored in the same CA room (William Gerling, personal communication).

Studies reported here were designed to determine how P. expansum enters non-wounded fruit and what factors might contribute to differences in susceptibility among fruit from different orchards. We evaluated the effects of orchard source, inoculation method, and storage conditions on the incidence of blue mold decay. Initial results from some of these studies were reported previously (18,21).


Fruit Harvest and Experimental Design

Six blocks of Empire apples in commercial orchards in Orleans County were selected to provide fruit with a range of susceptibility to decay. The experimental design involved six orchards, three inoculation methods plus non-inoculated controls, and three different factors related to inoculation timing and storage conditions. Individual trees within each orchard were used as replicates. Each postharvest treatment involved five apples per replicate (i.e., the smallest experimental unit was five apples), and each treatment combination was replicated five times. The experiment was repeated using fruit from the same tree rows within the same orchards in two successive years.

Fruit used for the experiments were harvested on 2 October 1997 and on 23 September 1998. In both years, the harvest window for long-term storage of the cultivar had just started (3) at the time samples were collected. In each orchard, samples were taken from every fourth tree along a single row until five trees had been sampled. Fruit from each tree were held separately and handled as replications so that any tree-to-tree differences in fruit maturity, mineral content, or physiology would be consistent throughout the replication. Fruit on the designated trees were selected arbitrarily from that portion of the tree canopy that was within easy reach (1 to 2 meters above ground), and samples included fruit from all quadrants of each tree. Fruit with visible defects and unusually large or small fruit were excluded from the samples.


Inoculation Method

Within several hours of harvest, apples from each orchard were arranged on spring cushion trays for the controlled inoculations. Four different inoculation treatments were applied using separate trays of 25 apples (five fruit per replication, five replications per orchard) for each inoculation method and for each orchard. For two of the inoculation treatments, micropipettes were used to place 10 l droplets of a spore suspension on the ends of stems (Fig. 1). Stems were left intact as they came from the orchard for the first treatment. For the second treatment, the ends of stems were snipped with a wire cutter just below the swelling at the end of the stem where the apple had been attached to the tree, and the 10 l droplets were then applied to the freshly cut stems. For the third inoculation method, the ends of the stems were sealed with silicon caulk to prevent spores from contacting the vascular bundles in the stem ends, and 0.5 ml of spore suspension was then pipetted into the stem cup of the apple. The fourth treatment involved non-inoculated control fruit arranged on spring cushion trays and handled just as inoculated fruit except that stems were not cut for any of the control fruit.



A
 
B

Fig. 1. Empire apple with 10 l droplet of spore suspension on the stem as it appeared immediately after inoculation (Fig. 1A) or with silicone caulk on stems and 500 l spore suspension deposited in the stem cups (Fig. 1B).


Spore suspensions were prepared by washing 7-day-old cultures of a benzimidazole-resistant isolate of P. expansum (P-132) with sterile distilled water. Based on hemacytometer counts, the initial spore suspensions were adjusted to provide final spore suspensions containing either 20,000 or 50,000 conidia/ml. The suspension with the higher spore density was used for stem-end inoculations, and the 10 l droplets delivered approximately 500 spores per stem. The lower-density spore suspension was used for the stem cup inoculations, so 0.5 ml provided 10,000 spores per stem cup. Spore suspensions were prepared the day prior to the first inoculations and were held in ice until used. The same spore suspensions were used for all inoculations in a given year.

Following inoculation, fruit were allowed to dry for about an hour. After an hour, the droplets on stems had dried and/or been absorbed into the stem vascular tissue. Fruit inoculated on the stem-ends were turned on their sides and trays were stacked four deep in the wooden storage boxes. Fruit inoculated in the stem cups were left upright to avoid spilling the remaining inoculum from the stem cups. Trays of fruit that received different inoculation treatments were randomized within the storage boxes so that the same treatments were not consistently on the top, bottom, or middle layers of the storage boxes.


Inoculation Timing/Storage Regime

In 1997, three trays were prepared for each orchard and inoculation treatment described above, and each tray was used for a separate inoculation timing/storage regimen. One regimen involved immediate inoculation and refrigeration (within 4 to 6 h of harvest) and establishment of CA atmosphere by nitrogen injection into the storage room within four days of inoculation. The second regimen was the same as the first, but fruit were held at 1.1C in air storage instead of going into CA storage. The third regimen involved holding fruit at ambient temperature (18 to 21C) for 20 to 24 h after harvest, then inoculating, refrigerating, and holding in CA storage as described for the first inoculation/storage regimen. Stem cutting for the part of this group that received the cut stem inoculation treatment was done about 15 h prior to inoculation. The same procedures were used in 1998, except that air storage was evaluated using fruit from only two of the six orchards.

In 1997, the storage atmosphere was adjusted to less than 5% oxygen within four days of harvest. The storage room was maintained at 1.1C, 1.6% oxygen and 2.2% carbon dioxide until the room was opened on 2 July 1998. Fruit were then held at 1.1C in air until they were evaluated for decay on 14 July. In 1998, similar CA conditions were established within four days of harvest, fruit were held in CA until 22 April 1999 and were then held in air storage at 1.1C until the incidence of decay was assessed on 14 May. In 1997, fruit held in air storage were evaluated and discarded on 27 May whereas in 1998 fruit held in air storage were evaluated on the same date as fruit stored in CA.


Fruit Maturity Assessments and Mineral Analyses

In both years, subsamples of 10 fruit from each replicate were used to determine fruit firmness and starch-iodine index at harvest, and the same 10 fruit were used to determine fruit mineral content. Fruit firmness was measured on opposite faces of each fruit using a Model EPT-1 Electronic Pressure Tester (Lake City Technical Products Inc., Kelowna, BC, Canada) fitted with an 11.1-mm tip. The starch-iodine index was determined using the method described by Blanpied and Silsby (3). To sample for fruit mineral content, a 6-mm slice was removed from each apple by cutting the apple perpendicular to the core. Two plugs 8 mm in diameter were removed from just beneath the skin on opposite sides of the slice using a cork-borer (27). The samples from 10 apples were combined, freeze dried, ground, and analyzed by atomic absorption at the Cornell ICP Tissue Analysis Lab (8). Field consultants for the six growers cooperating in the experiment provided results of leaf mineral analyses that had been conducted by the Cornell ICP Tissue Analysis Lab using bulked samples from each block.


Statistical Analyses

Analysis of variance was performed using the software program "SuperAnova" (Abacus Concepts, Inc., Berkeley, CA). Three-way factorial analyses were employed to compare effects of six farms, three inoculation methods, and two (1998-1999) or three (1997-1998) inoculation/storage regimes on incidence of fruit decay (Table 1). A similar six-farm by two-year factorial analysis was used to evaluate effects of orchard source (Table 2). Simple means or means within categories were then compared by applying the appropriate LSD tests.


Table 1. Percent of Empire fruit that developed decay during storage as affected by inoculation method and storage regime.

Inoculation
method
Inoculation/storage regimen
Cold air
storage
x
Controlled-atmosphere storagey
Inoculated
4-6 h after harvest
Inoculated
20-24 h after harvest
1997-1998
Stem end  <1 az 47 c 27 b
Cut stem 19 b 18 b 47 c
Stem cup   0 a   1 a <1 a
1998-1999
Stem end 0 30 b 31 b
Cut stem 0 48 c 56 c
Stem cup no data   1 a <1 a

 x Fruit in cold-air storage were evaluated after 34 weeks in 1997-1998 and after 33 weeks in 1998-1999.

 y Fruit in controlled-atmosphere storage were evaluated after 41 weeks in 1997-1998 and after 33 weeks in 1998-1999.

 z Means are from observations of 150 inoculated fruit (5 replicates 5 fruit per replicate 6 farms) except that only two farms were tested in air storage in 1998-1999. For statistical analyses, data were subjected to the angular transformation. Means separations were determined using LSD to compare means from the factorial analysis of 6 farms 3 inoculation methods 3 inoculation/storage regimens in 1997-1998 or 2 inoculation/storage regiments in 1998-1999. The means for cold-air storage in 1998-1999 were derived using fruit from only two farms and therefore these data were not included in the statistical analysis. Means within years and within columns that are followed by the same letter are not significantly different (P ≤ 0.05).


Table 2. Percent of Empire fruit that developed decay during controlled-atmosphere storage following inoculation of uncut stems as affected by orchard source and inoculation timing.

Orchard
source
Inoculated
4 to 6 h after harvest
Inoculated
20 to 24 h after harvest
1997-98 1998-99 Grand means
for 2 yr
1997-98 1998-99 Grand means
for 2 yr
A   60 bx    64 c       62 c 36 a   56 c       46 b
B   44 ab    60 c       52 bc 44 a   52 bc       48 b
C   24 a    48 bc       36 ab 16 a   28 abc       22 a
D   44 ab    28 ab       36 ab 28 a   12 a       20 a
E   68 b      8 a       38 ab 40 a   20 ab       30 ab
F   36 ab    12 a       24 a 20 a   52 bc       36 ab

 x Means for each year are from observations of 25 fruit. Data were subjected to the angular transformation for statistical analyses, but arithmetic means are shown. Means separations were determined using LSD to compare transformed means from the factorial analysis of 6 farms 2 years of observations. Means within columns followed by the same letter are not significantly different (P ≤ 0.05).


Linear regression analyses were performed using the Microsoft Excel (Excel:Mac v.X, Microsoft, Inc., Seattle, WA). Only a single composite analysis of leaf mineral content was available for each of the six orchards in each of the two years (i.e., N = 12), so relationships between leaf mineral content and decay incidence were evaluated by comparing leaf mineral content for each orchard and year to the corresponding within-orchard means for incidence of decay. The latter was derived from the five replicate fruit samples taken from each orchard in each year. With-in orchard means were also used for regression analyses involving effects of fruit mineral content on incidence of decay.


Results: Decay Incidence After Storage

When results from all six orchards were combined, decay incidence in CA-stored, stem-inoculated fruit with intact stems ranged from 27 to 47% over the two years whereas the incidence of decay for similar fruit held in cold-air storage was less than 1% (Fig. 2) (Table 1). Results for fruit with cut stems were similar except that decay incidence of fruit in cold-air storage reached 19% in one year. Placing a spore suspension in the stem cup never caused more than 1% decay (Table 1). Comparative susceptibility of intact stems and cut stems varied with year and inoculation timing. For fruit harvested in 1997, the non-inoculated control fruit with intact stems from CA storage had 0 and 0.7% of fruit with decay for fruit inoculated 4 to 6 and 20 to 24 h after harvest, respectively. Comparable numbers for fruit harvested in 1998 were 1.3 and 8.0%, respectively. The reason for the high incidence of decay in control fruit held for 20 to 24 h after harvest in 1998 is not known.


 

Fig. 2. Empire apples with early stages of blue mold decay that developed when P. expansum invaded fruit via stems during CA storage.

 

Orchard source affected susceptibility to decay following inoculation of intact stems, but the ranking of the various orchards was not consistent from year to year (Table 2). Orchards A and B had the highest mean incidence of decay over two years for both immediate and delayed inoculations. Orchards C and D had moderate levels of decay in both years. Orchards E and F showed the greatest variation from year to year.

Fruit maturity, as measured by starch index and fruit firmness at harvest, varied among the orchards (Table 3). However, starch index and decay incidence showed no linear relationship whereas decay incidence was correlated with fruit firmness (R2 = 0.343; P = 0.045).


Table 3. Starch-iodine indices and flesh firmness of Empire apples from the six orchards used in 1997 and 1998. In 1998, fruit firmness was also evaluated at the end of the experiment after fruit were removed from controlled-atmosphere storage.

Orchard
source
1997x 1998x
Starch
iodine
index
y
Fruit
firmness

(Newtons)
Starch
iodine
index
y
Fruit
firmness

(Newtons)
Fruit
firmness
(Newtons)
after storage
A     3.6 a     78.4 d 4.5 b    72.6 bc 70.5 b
B     3.6 a     74.5 ab 2.6 a    74.8 c 64.1 a
C     5.5 d     75.8 bc 2.7 a    70.5 b 69.3 b
D     4.0 ab     74.0 ab 3.2 a    67.3 a 63.7 a
E     4.5 bc     76.6 cd 3.1 a    68.9 ab 63.9 a
F     5.1 cd     72.9 a 3.3 a    72.2 bc 65.5 a

 x Means from 10 fruit per replicate and 5 replicates per orchard. Mean separations were determined using Fishers Protected LSD. Means within columns followed by the same small letter are not significantly different (P ≤ 0.05).

 y The starch-iodine index (reference 2) ranges from 1 for immature fruit to 8 for mature fruit with no residual starch. Harvest of Empire fruit for long-term storage is recommended when the starch-iodine index values are between 4.2 and 4.6, but commercial harvest is sometimes initiated before apples reach 4.2.


Boron concentrations in leaves and fruit and phosphorus content of leaves showed highly significant correlations with decay incidence (Table 4). Boron concentrations in fruit were also positively correlated with both fruit firmness and with phosphorus content of fruit.


Table 4. Relationships between incidence of decay in inoculated fruit with intact stems (CA storage) and various factors that might affect susceptibility to decay as observed for six orchards over two seasons (N = 12).

Factors evaluated as predictors
of decay susceptibility
Probability of linear significance (P-value) R2
Boron concentration in leaves 0.0013 0.661
Boron concentration in fruit 0.0025 0.615
Phosphorus concentration in leaves 0.0099 0.502
Phosphorus concentration in fruit 0.0299 0.390
Calcium concentration in leaves 0.0862 0.266
Calcium concentration in fruit 0.5692 0.033
Manganese concentration in leaves 0.8870 0.002
Manganese concentration in fruit 0.4939 0.048
Nitrogen concentration in leaves 0.8230 0.005
Potassium concentration in leaves 0.3320 0.094
Magnesium concentration in leaves 0.4570 0.056
Zinc concentration in leaves 0.8580 0.003
Copper concentration in leaves 0.5558 0.036
Starch-iodine index 0.8340 0.005
Fruit firmness 0.0450 0.343
Other factors
Fruit boron as a predictor of fruit phosphorus 0.0069 0.535
Fruit boron as a predictor of fruit firmness 0.0389 0.361
Fruit phosphorus as a predictor of fruit firmness 0.3077 0.104
Fruit boron as a predictor of leaf phosphorus 0.0065 0.540
Fruit boron as a predictor of fruit calcium 0.8730 0.002

Discussion

Results from this study showed that P. expansum can cause decays of non-wounded Empire apples by growing down through fruit stems during long-term CA storage. Stem-end invasion of pears by Penicillium expansum and Botrytis cinerea has been reported previously (15,24), but this is the first report of P. expansum invading apples through stems under commercial storage conditions. Benzimidazole-resistant inoculum can be deposited on stems of freshly harvested fruit via recycling drench solutions used to apply DPA and fungicide immediately after harvest. The fact that spores deposited in stem cups did not initiate decay is consistent with another recent report where mechanically harvested apples failed to develop decay when inoculum was applied to the stem cup (10).

Although stem-inoculated apples developed stem-end decay during CA storage, decay did not develop in similarly inoculated fruit held in air storage except for 1997 fruit with cut stems. However, we cannot conclude from these trials that stem invasion occurs only under CA conditions. The air-stored fruit were transported from western New York to the Hudson Valley Lab after inoculation and were not placed into cold storage for 6 to 10 h after inoculation whereas the CA fruit were placed into cold storage immediately after inoculation. Relative humidity in the storage rooms was not monitored, but the size and design of the storage rooms resulted in maintenance of higher relative humidity in the large CA room than in the small cold-air storage room. Thus, several factors other than storage atmosphere may have contributed to differences observed for fruit in CA versus cold-air storage.

Packinghouse operators have reported that stem-end decays become evident in stored fruit only after about 6 mo of CA storage. Many of the inoculated fruit that did not develop decay in these experiments had stems that were partially browned (Fig. 3), suggesting that P. expansum had invaded the stem but had not yet grown far enough to reach the fruit. Stems on stem-inoculated fruit that developed decay were uniformly brown. Thus, it appears that fruit stems act almost like a slow-burning fuse and that stem-end decay is initiated only after P. expansum has succeed in growing through the length of the stem. Stem end decays in air-stored fruit are unlikely to become a commercial problem because most air-stored fruit are marketed and consumed within 3 months of harvest, long before P. expansum can grow through the length of the stem.


 

Fig. 3. Stem-inoculated Empire apple that failed to develop decay during CA storage but showing a sharp line of demarcation between living and dead tissue in the stem.

 

The effect of cutting stems was investigated to determine if freshly cut surfaces would prove more susceptible to invasion by P. expansum than intact stems where the natural abscission layer might play a role in preventing infection. Other fruit were also inoculated 20 to 24 h after harvest to determine if a short holding period after harvest would influence susceptibility of stems to infection. The 24-h delay did not reduce infection incidence, so the timing of inoculation within 24 h after harvest does not appear critical to the infection process. Holding fruit longer prior to refrigeration could result in loss of fruit quality due to fruit softening at warmer temperatures. Results reported here for apples are consistent with a previous report showing that pear stems remained susceptible to infection by P. expansum after 2 days at 20C or 6 months at -1C (24).

The fact that cut stems developed more decay in three of the four comparisons (Table 1) may indicate that the abscission layer in intact stems plays a role in slowing infection, or it may have occurred because stems were slightly shorter after cutting and therefore P. expansum could traverse the stem more quickly. Understanding why cut stems allowed a higher incidence of decay is not critical because our results show that cut or broken stems are not required for P. expansum to gain access to stem tissue.

Susceptibility of fruit to decay via stem invasion was highly correlated with both boron and phosphorus concentrations in fruit, but we suspect that the relationship with boron is the most important factor for several reasons. First, the regression coefficients for effects of fruit and leaf boron concentrations on incidence of decay were higher than those for phosphorus. Second, among the six orchards, Orchard E had the highest leaf boron levels and the most decay in 1997 whereas in 1998 Orchard E had nearly the lowest boron concentrations in fruit and it also had the least decay (Fig. 4). Phosphorus levels for this orchard varied little from year to year. Third, in contrast to boron, phosphorus has not been implicated in disease susceptibility in apples. Hansen (9) reported that low boron concentrations in leaves (10 to 15 g/g) were associated with increased incidences of leaf spotting in Coxs Orange Pippin apples and that concentrations above 30 g/g resulted in discoloration and brittleness of leaves. Wojcik et al. (30,31) noted that post-bloom boron sprays applied to Elstar apple trees resulted in increased susceptibility to Gloeosporium fruit rot but not Penicillium infection, whereas pre-bloom boron sprays applied to Sampion apples resulted a lower incidence of Gloeosporium fruit rot during storage. Lee and Kim (13) reported that Fuji apples receiving high rates of soil-applied boron produced fruit that matured earlier and were more susceptible to Macrophoma rot. In our experiments, fruit firmness showed a significant positive correlation with fruit boron (r2 = 0.36), an effect that is exactly opposite of what would be expected if high boron triggered early ripening.


 

Fig. 4. Effect of boron concentrations in leaves (upper graph) and fruit (lower graph) on susceptibility of fruit to postharvest decay as determined using means for all fruit with intact stems that were inoculated within 24 h after harvest and held in CA storage. Labels beside data points designate orchard source and year of harvest (1997 or 1998).

 

This is the first report of a relationship between high boron concentrations and susceptibility of apples to decay caused by P. expansum. The fact that calcium concentrations in apples can affect development of postharvest decays and physiological disorders is well-documented (4,5,6,14,26), but in this study we did not find any relationship between fruit calcium content and susceptibility of apples to stem-end invasion. The relationship between high boron levels and increased susceptibility to decay deserves further attention. If the relationship can be verified in additional well-designed tests, then growers may want to reduce boron applications in Empire blocks where foliar boron levels exceed 35 ppm. Alternatively, analysis of foliar boron levels in late summer might be used as a predictor for postharvest decay problems and fruit from high-boron orchards could be marketed earlier in the season.


Acknowledgments

This work was funded in part by grants from the New York State Apple Research and Development Program and the New York Apple Research Association, and by Smith Lever funds from the Cooperative State Research, Education, and Extension Service, US Department of Agriculture. The authors thank Jackie Nock for technical assistance. Thanks to George Lamont, David Kast, Bruce Kirby, Robert and Eric Brown, and Orren and Gary Roberts for supplying Empire fruit for experiments reported here. A special thanks to Bill Gerling and his associates at Lake Ontario Fruit for their assistance in executing some of the experiments, and to Randy Paddock and Jim Misiti for assisting in fruit harvest.


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