Washington Tree Fruit Research Commission

Research Reports

New approaches to decay control of pear (2008)

FINAL PROJECT REPORT
WTFRC Project #
YEAR 0/0
Organization Project #
Title:New approaches to decay control of pear
PI:Robert A.Spotts
Organization:OSU Mid-Columbia Ag Research and Extension Center Telephone/email: 541-386-2030 ext. 15/ robert.spotts@oregonstate.edu
 PDF version of report

Cooperators:

WSU (Chang-Lin Xiao)

SOREC (David Sugar)

Ag Canada (Peter Sholberg, Dan O’Gorman)

New Zealand HortResearch (Trish Virgin, Monika Walter)

Lincoln University (Alison Stewart)

Significant findings

·         A 4-factor gray mold risk prediction model has been developed for the major Oregon and Washington pear districts.

·         A real time PCR method to determine the concentration of decay spores (Penicillium expansum and Mucor piriformis) in dump tank and flume water has been developed.

·         758 new bull’s-eye rot isolates have been identified. N. perennans  is the most common species in Yakima and Wenatchee and N. alba the most common in Hood River and Medford.

·         A new unnamed species of bull’s-eye rot (Neofabraea sp. nova) was found in all major pear districts

·         The benzimidazoles thiophanate methyl (Topsin) and thiabendazole (Mertect) appear to have the most effect on N. alba and N. perennans

·         A qPCR method was developed to determine threshold “residues” of the biocontrol agent CIM required on pear fruit for optimum decay control.

·         The most effective preharvest spray for overall decay control in 2005-6 was a tank mix of Topsin M and Nutraphos 24.

·         Postharvest fungicides Pristine, Penbotec, and Scholar controlled blue and gray mold·         Muscodor albus, a biological control agent, significantly controlled blue mold, gray mold, and mucor rot of d’Anjou pear fruit.

•     Protective Chemistries paint formulations 1020, 1024, 3020, and 4020 were very effective for prevention of growth of Botrytis cinerea, Mucor piriformis, and Penicillium expansum on wood and plastic surfaces.

Results and discussion

1.      New model for decay risk prediction A 4-factor gray mold risk prediction model is shown below in Table 1.  The model was developed using data from pear orchards in Oregon, Washington, and New Zealand in 2004-6 (Table 2).  Actual decay aligned extremely well with the predicted risk level for fruit from all districts in all three years (Table 2).          

Model validation on a larger scale is necessary before the model can be used by the fruit industry.  During the 2007-8 season, the model is being validated in orchards in Wenatchee (Dr. Chang–Lin Xiao), Medford (Dr. David Sugar), and the Mid-Columbia (Dr. Bob Spotts) and will use packinghouse cull analyses when available. 

Improvements are continually being made and include new, more specific PCR primers for DNA analysis, use of Millipore membranes for high efficiency capture of spores, and scale-up oflaboratory methodology to handle large sample numbers necessary for commercialization.
 

Table 1.  Pear gray mold risk prediction model (version 1.00)
  Orchard rating4
DNA1Fungicide2Rain3123
LYesNoLLM
LYesYesLMH
LNoNoLMH
LNoYesMHE
HYesNoLMH
HYesYesMHE
HNoNoMHE
HNoYesHEE

1L = B. cinerea DNA 0 to 2.2 pg/cm2; H = over 2.2 pg/cm2.

2,3Yes = fungicide applied within 4 weeks of harvest; 0.02 inches within 2 weeks of harvest.

41 = young to moderate age trees, excellent horticultural and pest/disease practices.

42 = moderate age trees, average horticultural and pest/disease control practices.

43 = old trees, poor horticultural and pest/disease control practices.

5Risk levels: L = low, M = moderate, H = high, E = extreme. 

Table 2.  Pear gray mold predicted risk vs. actual decay in cold stored fruit from Oregon,  Washington, and New Zealand
 2004-20052005-20062006-2007
 OrchardxPredicted riskyGray mold (%)zPredicted riskyGray mold (%)zPredicted riskyGray mold (%)z
1E14.0H8.7H7.4
2H8.0H7.3H3.3
3H7.0H6.6H3.0
4H5.9M5.9M3.1
5H4.2M5.1M1.1
6M2.6M3.8M0.9
7M2.2M3.4M0.6
8M2.2L2.1------
9 Medford    M1.3
10 Medford    M1.0
11 Medford    M0.7
12 Wenatch    MND
13 Wenatch    MND
14 Wentach    LND
15 NZM1.4L0.7  
16NZL1.0L0.5  
17NZL0.7L0.4  
18NZL0.3L0.3  
19NZL0.3L0.3  
20NZL0.1L0.1  
xOrchards 1-8 in Mid-Columbia; Orchards 9-11 in Medford; Orchards 12-14 in Wentachee; Orchards 15-20 in Motueka, New Zealand.
yRisk levels: L = low, M = moderate, H = high, E = extreme.

zDecay after 6 months storage at 30°F.

 The average incidence of gray mold at each risk level varied from year-to-year (Table 3).  This appears to be related to yearly changes in susceptibility of fruit and matches the annual susceptibility index determined from a standardized lab test (Table 4).  For example, the percent gray mold was highest in 2005-6 and lowest in 2006-7 in both actual decay (Table 3) as well as in the standard test (Table 4).  
Table 3.  Amount of gray mold from decay model risk levels
 Risk levely
 LowModerateHighExtreme
YearPercent gray moldz
2004-50.52.16.514.0
2005-60.64.57.5---
2006-7---1.33.7---
yRisk levels from gray mold risk prediction model.
zGray mold is average of all orchards in each risk level.
 
Table 4. Susceptibility of Anjou pear fruit to decay in standardized laboratory conditions
 Infection indexz
YearGray moldBlue moldMucor rot
200422.7b28.2a48.4b
200528.0b28.0a58.2b
200613.0a31.4b49.2b
200732.9c26.5a21.6a

zIndex is calculated as lesion diameter (mm) x proportion of fruit infected. Numbers followed by the same letter within columns are not significantly different at P = 0.05.

2.   DNA techniques for rapid, accurate detection of decay spores in packinghouses             Excellent agreement was found between the amount of P. expansum DNA in “spiked” dump tank water from three packinghouses and the spore counts from traditional dilution plates.  A new method was introduced in 2007 using a Millipore membrane rather than centrifugation to remove spores from the water prior to DNA extraction.  The new method is much faster and more accurate than the old method.  The experiment needs to be continued with additional water samples from Oregon and Washington packinghouses.  

 Relationships between decay and spore loads in water

Mucor rot and blue mold decays are closely related to spore loads in packinghouse water systems.  We found that the relationship is similar for Bosc pear in 2006(solid line), Bosc in 2007 (dashed line), and Anjou in 2007 (dotted line).  The steep curve for Mucor rot between 0 and 500 spores per ml of water indicates that reduction of spore numbers in this part of the curve will result in significant reductions in Mucor rot.For blue mold, the curve increases more gradually than for Mucor rot, and reductions between 0 and 1,000 spores per ml of water will result in gradual decreases in the amount of decayed fruit.These results emphasize the importance of good sanitation in the packinghouse.  Spore loads in packinghouse water should be reduced as much as possible to reduce decay in storage. 

3.      Bull’s-eye rot species in Washington and Oregon and fungicide sensitivity

We have identified 758 isolates of Neofabraea from decayed pear (Table 5) and apple (Table 6) fruit.   N. perennans  is the most common species in Yakima and Wenatchee and N. alba the most common in Hood River and Medford.  N. malicorticis was not found in any Oregon or Washington orchards but is known to occur on the west side of the Cascade Mountains.  The new, unnamed species of Neofabraea was found in all four districts and was most common on apples from Yakima and Wenatchee (Xiao collection). 

Table 5. Summary of Neofabraea (bull’s-eye) collection in pears 
 Percent of total
LocationN.albaN.perennansNew speciesNumber samples
Yakima0.0100.00.020
Wenatchee15.884.20.0101
Mid Columbia64.634.90.6175
Medford78.216.05.8312
 
Table 6.  Summary of Neofabraea (bull's-eye) collection in apples
 Percent of total
LocationN.albaN.perennansNew speciesNumber samples
Yakima0.081.518.527
Wenatchee0.088.511.5113
Mid Columbia0.00.00.00
Medford90.00.010.010



 
Table 7.  Summary of Neofabraea (bull's-eye) entire apple and pear collection
                            Percent of total
LocationN.albaN.perennansNew speciesNumber samples
Yakima0.089.410.647
Wenatchee7.586.46.1214
Mid Columbia64.634.90.6175
Medford78.615.55.9322

 Effect of fungicides on Neofabraea alba and N. perennans in vitro

In order to achieve satisfactory control of bull’s-eye rot, it is critical to know the Neofabraea species profile in each district and use fungicides that have good activity against those species.

We found that the four most effective fungicides for bull’s-eye rot control on all four species of Neofabraea were the two benzimidazoles, Mertect and Topsin M, Penbotec (pyrimethanil), and Pristine (pyraclostrobin + boscalid) (Table 8).  In addition to these four fungicides, other fungicides gave acceptable control of some species of Neofabraea but not other species.  For example, Scholar controlled bull’s-eye rot caused by N. malicorticis but not by the other three species. 

Copper and ziram have been used routinely for many years in the Pacific Northwest for control of Neofabraea cankers on trees and bull’s-eye rot on fruit.  We found that ziram was moderately effective, but copper gave poor control of all species except N. malicorticis (Table 8).   

Table 8.  Control of bull’s-eye rot of d’Anjou pear fruit with postharvest dip of fungicides
  Percent bull’s-eye rot caused byx
Treatment    RateyN. perennansN. albaN. sp. novaN. malicorticis
Penbotec 400 SC1000 ppm0.0a0.0a1.7ab0.0a
Penbotec 400 SC500 ppm3.3ab0.0a4.2b0.0a
Topsin M 70WP1.0 lb/200 gal0.8ab1.7ab0.0a1.9a
Mertect 340F16 oz/100 gal0.8ab7.4bc2.6a0.0a
Pristine 38%2000 ppm7.5b0.0a0.8a0.0a
Procure 480SC12 oz/200 gal85.8d13.3c3.3a23.4cd
Sovran 50WDG6.4 oz/200 gal80.8d29.0d18.0b4.2ab
Ziram 76DF8.0 lb/200 gal66.1c55.8ef22.4b18.6cd
Scholar 230 SCz300 ppm68.328.358.30.0
Scholar 230 SC150 ppm91.7ef44.2de82.5d9.4bc
Flint 50WDG2.5 oz/200 gal90.6de67.5f32.5b19.3bc
Cuprofix Ultra 4016 lb/200 gal96.4fg98.1g63.5c26.9d
Pencozeb 75DF3.0 lb/200 gal93.9ef69.5f60.8c45.6e
Water control---100.0g97.4g79.2d68.0f
xNumbers within columns followed by the same letter are not different according to protected LSD test at P = 0.01.
yRate per 200 gal is the per acre rate.
zSingle trial results, no statistics.

        4.      Use of qPCR to determine “residues” of a biocontrol agent on pear fruitThe recommended concentration of the biological control yeast CIM for decay control is 2 x 108 cfu/ml.  When Anjou (solid lines for 2006 and 2007) and Bosc (dashed lines for 2006 and 2007) fruit were treated with this concentration, the amounts of DNA on the fruit surfaces were about 1700 and 460 ng per cm2, respectively.  This method can be used to assure that CIM is being properly applied to pear fruit on the packing line or in the drench and will result in optimum decay control.  Negotiations are underway to license CIM and obtain EPA registration. 

5.      Preharvest and postharvest fungicides for decay control

Preharvest treatments.  For blue mold control, two treatments (Ziram and the foliar nutrient Nutraphos) were ineffective.  The most effective preharvest spray for blue mold was Topsin M, either alone or combined with Nutraphos 24 or Ziram.  All fungicides controlled gray mold, but the foliar nutrient Nutraphos was not effective.  Pristine was effective when applied twice but not as a single application. 

Table 9.  Preharvest fungicides to control postharvest decay of d’Anjou pears
  Blue mold (%)yGray mold (%)y
Preharvest treatment and rate/APHIz (wk) 2004 2005 2006 2004 2005 2006
Control--23b36d23b9c9b3ab
Pristine 38 WG 14.5 oz28a------7bc------
Pristine 38 WG 14.5 oz15a35d---3ab9b---
Pristine 38 WG 14.5 oz 2+1---23c10a---3a1a
Topsin M 70WSB 1 lb210a25c9a1a2a3ab
Topsin M 70WSB 1 lb18a11b10a2a2a2a
Ziram 76DF 8.0 lb  219b45e22b2ab2a6b
Nutraphos 24 15 lb2---33d23b---8b5b
Topsin M 70WSB 1 lb + Nutraphos 24 15 lb 2 --- 3a 8a --- 2a 1a
Topsin M 70WSB 1 lb + Ziram 76DF 8.0 lb 2 --- --- 8a --- --- 2a
Ziram 76DF 8.0 lb  + Nutraphos 24 15 lb 2 --- --- 24b --- --- 1a
yNumbers followed by the same letter within columns are not significantly different at P = 0.05 according to ANOVA and protected LSD of square root transformed data.
zPHI =  preharvest spray interval in weeks.
 Postharvest treatments - Drench application (Table 10 ).  Control fruit (water drench) had 2.7% blue mold.  All Pristine and Penbotec drench treatments significantly reduced blue mold and gave 100% control.  Ethoxyquin increased blue mold, probably by acting as a wetting agent to increase penetration of spores into wounds.  Gray mold in control fruit was 13.1%, and ethoxyquin was not significantly different from the control.  Pristine and Penbotec gave 100% control of gray mold.  Ethoxyquin caused slight phytotoxicity, and Pristine at 2000 ppm caused very slight phytotoxicity.  Phytotoxicity appeared as dark spotting at lenticels where fruit was in contact with the polyliner and was slow to dry.  
Table 10.  Control of decay of d’Anjou pear fruit with postharvest drenches of Pristine at MCAREC, Hood River, OR in 2006-7
  Percent fruit infectedz
 Treatment Rate product per 100 galBlue mold Gray mold
Pristine 2000 ppm70.0 oz0.0a 0.0a
Pristine 1000 ppm35.0 oz0.0a 0.0a
Pristine 500 ppm17.5 oz0.0a 0.0a
Pristine 250 ppm8.75 oz0.0a 0.0a
Penbotec 1000 ppm1.0 quart0.0a 0.0a
Ethoxyquin 2700 ppm2.0 quarts9.5c 14.7b
Water control---2.7b 13.1b
zNumbers followed by the same letter within columns are not significantly different at P = 0.05.

 Postharvest treatments – Line spray application (Table 11 ).  Water and wax control fruit had 27.5 and 5.8% of wounds infected with blue mold, respectively.  The wax contains morpholine, which probably contributed to the reduction in decay. All rates of Pristine in both water and wax gave excellent control of blue mold, and there were no significant differences among rates.  Penbotec in water and wax also gave excellent control of blue mold.

Gray mold incidence was 35.8 and 8.3% for the water and wax controls, respectively. All rates of Pristine in both water and wax gave excellent control of gray mold, and there were no significant differences among rates.  Penbotec in water and wax also gave excellent control of gray mold.  In wax, the Penbotec treatment had significantly less gray mold than the lowest rate (250 ppm) of Pristine. No phytotoxicity was observed with any of the treatments in water or wax.   

Table 11.  Control of blue mold and gray mold of d’Anjou pear fruit with postharvest line spray application of Pristine at MCAREC, Hood River, OR in 2006-7
  Percent wounds infectedy
TreatmentzRate (ppm a.i.)Blue moldGray mold
1. Water control---27.5c35.8d
2. Pristine2500.0a0.2ab
3. Pristine5000.2a0.5ab
4. Pristine10000.0a0.0a
5. Penbotec10000.2a0.0a
6. Wax control---5.8b8.3c
7. Pristine2500.0a0.7b
8. Pristine5000.0a0.2ab
9. Pristine10000.2a0.4ab
10.Penbotec20000.0a0.0a
yNumbers followed by the same letter within columns are not different at P = 0.05.
zTreatments 2 to 5 are water suspensions, treatments 7 to10 are wax suspensions.


 6.      Evaluation of Muscodor for decay control

The biological control fungus M. albus significantly controlled blue mold, gray mold, and mucor rot of d’Anjou pear fruit at 30° F to 41° F.  Phytotoxicity needs to be reduced before M. albus can be used commercially on pear fruit.  As storage temperature was reduced, phytotoxicity decreased.  No phytotoxicity was observed at the 4 gram per box rate at 30° F.   

Table 12.  Control of pear decays with Muscodor albus in 2004-2005
 

Percent gray moldy

Percent mucor roty

Percent blue moldy

Ratez41° F32° F30° F41° F32° F30° F41° F32° F30° F
097b97b100c100c100c100c100c100c100d
0.56a50a72b72b39a50b61b89bc78bc
16a47a31a70b58b45b19a84b53a
23a56a67b51b61b22a14a55a69ab
43a56a53ab26a53ab8a28a78b89cd
yNumbers followed by the same letter within columns are not significantly different at P = 0.05 according to least significant difference test.
zRate of M. albus grams per liter.

 7.         Evaluation of paint formulations for control of apple and pear postharvest decay fungal pathogens on plastic and wood surfaces

Paint formulations (Protective Chemistries 1020, 1024, 3020, and 4020) were very effective for prevention of growth of Botrytis cinerea, Mucor piriformis, and Penicillium expansum on wood and plastic surfaces.  Mortality of spores of these fungi was 100% in most time/temperature combinations on wood and plastic.  Use of these paints on apple and pear bins and on surfaces in packinghouses and cold storage rooms will significantly reduce or eliminate spores and growth of several important postharvest fungal pathogens and may be a key component in an integrated decay control program. 

Table 13.  Effect of three paint formulations on sporulation and growth of Botrytis cinerea on wood and plastic chips
   0°C
 1 month @ 20°C2 months3 months
TreatmentCfu/chipRatingyCfu/chipRatingyCfu/chipRatingy
Plastic
30200az0.0a0a0.0a0a0.0a
Unpainted845b2.0b198a1.1b344a1.1b
Wood
10200a0.0a0a0.0a0a0.0a
40200a0.0a0a0.0a0a0.0a
Unpainted12a0.4a115a0.3a198a1.8c
yRating 0 = no fungal visible growth; 1 = some diffuse growth; 2 = moderate to heavy growth.zEach value is the mean of 3 replications with 3 chips per replication. Numbers followed by the same letter are not significantly different at P = 0.05 according to ANOVA and the protected least significant difference test.  Initial cfu/chip = 8,000. 
 




Table 14.  Effect of three paint formulations on sporulation and growth of Penicillium expansum on wood and plastic chips
   0°C
 1 month @ 20°C2 months3 months
TreatmentCfu/chipRatingyCfu/chipRatingyCfu/chipRatingy
Plastic
30200az0.0a0a0.0a0a0.0a
Unpainted89,246a0.7a2,908b0.3a25,666b1.1b
Wood
10203,595a0.0a0a0.0a0a0.0a
40200a0.0a0a0.0a0a0.0a
Unpainted56,343a0.2a175a0.9b6,950a1.4b
yRating 0 = no fungal visible growth; 1 = some diffuse growth; 2 = moderate to heavy growth.zEach value is the mean of 3 replications with 3 chips per replication. Numbers followed by the same letter are not significantly different at P = 0.05 according to ANOVA and the protected least significant difference test.  Initial cfu/chip = 7,000 at 20°C, 10,000 at 0°C. 
  
Table 15.  Effect of paint formulations 1020 (New) and 1024 on sporulation and growth on wood of three decay fungi of pear fruit, OSU MCAREC, Hood River, 2007
  0°Cx
 1 month at 20°Cx2 months3 months
 CFU/chipRatingyCFU/chipRatingyCFU/chipRatingy
Penicillium expansum
1020 (New)ndz0a0a0a328a0a
1024nd0a0a0a297a0a
Controlnd2b355,402b2b1,814,302b2b
Mucor piriformis
1020 (New)0a0a8a0a0a0a
10240a0a8a0a0a0a
Control2,777,667a2b283,333b2b257,990b2b
Botrytis cinerea
1020 (New)nd0a0a0and0a
1024nd0a0a0and0a
Controlnd1b19a0.4bnd2b
xEach value is the mean of 3 replications with 3 chips per replication. Numbers followed by the same letter are not significantly different at P = 0.05 according to ANOVA and the protected least significant difference test.  Initial CFU/chip = 7,261 for P. expansum, 1,845 for M. piriformis, and 5,777 for B. cinerea.yRating 0 = no fungal visible growth; 1 = some diffuse growth; 2 = moderate to heavy growth.znd = contamination and/or sporulation not determined. 
 

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