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Cytospora Canker on Colorado Peaches: Current Research
Jane E. Stewart Plant Pathologist
Colorado State UniversityBioagricultural Sciences and Pest Management
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Cytospora canker• Caused by fungal species Cytospora• Opportunist pathogens, cannot invade healthy
intact bark
Pruning cut with infection
canker canker
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Disease cycle
Fungus persists and forms fruiting structures in dead wood and cankers
Conidia spread by rain; germinate and infect injured and dead tissues, expands as cankers
Humid, wet
Leaf scars, damaged buds
Crotches with narrow branch angles Shaded winter
damaged twig
Unhealed pruning stub
Poorly healed wound
Biggs and Grove 2006. Plant Health Instructor
Canker rings formed by infection and host callus formation
H . Larsen
H . Larsen
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Cytospora presence in Colorado
• 200 acres• 42 varieties• 2-32 year old orchards
(mean 11 years)• 100% of orchards surveyed
infected (mean 75% infected)• Ave number of infections per
tree was 5.2 (range 0-27)• Currently analyzing relationships
between practices and infection
Surveyed
Results
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Questions
• How many Cytospora species cause infections on peaches ?
• What chemicals are effective against Cytospora?– Preventative vs. suppressive?
• Are trees susceptible year around?
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Species differ biologically
• Three species have been identified on peach in other peach growing regions:– Cytospora leucostoma– Cytospora paraleucostoma– Cytospora cincta
• Variation in occurrence and virulence on different peach varieties
• Found on different locations of the tree
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What species are found at Orchard Mesa and Rogers Mesa?
• Collections made in July-August 2015– 135 isolates were recovered at different elevations – Identified species based on DNA sequencing
K. K im broughH . Larsen
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One species found on Colorado peach
• Cytospora leucostoma
• No difference between orchards low vs. high elevation
• No difference between winter/summer isolates
• Apple and peach isolates genetically distinct
Stewart et al. In prep
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Risks of other species introduced into Colorado
• Virulence – Difference among C. leucostoma, C. paraleucostoma,
C. cincta• Growth at different temperatures• Fungicide sensitivity• Sporulation• Differences with peach varieties
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Questions
• How many Cytospora species cause cankers and gummosis ?
• What chemicals are effective against Cytospora?– Preventative vs. suppressive?
• When are trees susceptible?
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What chemicals are effective against Cytospora?
• Evaluate the efficacy of conventional and organic fungicides for Cytospora leucostomacontrol
• Test wound sealing alternatives to develop preventive and spore suppressive approaches in existing orchards
C. leucostoma cankerInfected prune wound
Stephan Miller
Miller et al. Plant Disease. submitted
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Chemical Testing PhasesLaboratory Assay:• Testing chemicals in vitro on plates• Testing chemicals on detached branches
Field Trials:• Testing chemical sprays as preventive measure on
branches• Testing chemicals embedded in paints as
preventive measure on branches• Testing chemicals on existing cankers
for spore suppression
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Treatmentname
Active ingredientLabel rate (per 200
gal.)Rate
chosenMode of Action
MicrothiolDisperss Sulfur 10-20 lb 15 lb Multi-site
Fontelis Penthiopyrad 14-20 oz 17 oz RespirationTorino Cyflufenamid 3.4 oz 3.4 oz UnknownPristine Pyraclostrobin & Boscalid 10.5-14.5 oz 12 oz RespirationAliette WDG Fosetyl 10 lb 10 lb UnknownTopsin M WSB Thiopthanate-methyl 1-1.5 lb 1.25 lb Cytoskeleton/
motor proteins
Benlate WP Benomyl 24-32 oz 28 oz Cytoskeleton/ motor proteins
Captan N-Trichloromethylthio-4-cyclohexene-1,2-dicarboximide 3-4 qt 3.5 qt Multi-site
Inspire Super Difencoconazole &Cyprodinil 16-20 oz 18 oz
Protein synthesis
Ziram Zinc dimethyldithiocarbamate 3 - 5.3 lb 1.15 lb Multi-site
Conventional Chemicals Evaluated
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Treatmentname
Active ingredientLabel rate (per 200
gal.)Rate
chosenMode of Action
CaCl CaCl 48 oz 48 oz Multi-siteNeem Oil Neem Oil 3 qt 3 qt Not classifiedMpede Potassium salts 2-4 gal 3 gal. Multi-siteKaligreen Potassium bicarbonate 2.5-3 lb 2.75 lb Not classified
Serenade Bacillus subtilis 14-20 oz 17 oz Lipid synthesis/ transport
NuCop WP Copper Hydroxide 8-20 lb 10 lb Multi-site
Badge X2 Copper Hydroxide &Copper Oxychloride 3.5-5.25 lb 4.25 lb Multi-site
ZnSO4 ZnSO4 4-6 lb 5 lb Multi-siteLime sulfur Calcium polysulfide 20-24 gal. 22 gal. Multi-site
Organic Chemicals Evaluated
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2. Isolates inoculated onto plates
3. Inoculated plates incubated at25°C for 7 days
4. Colony areas assessed every 24 hours
MethodsIn vitro chemical plates
1. Chemical treatments amended in agar solution, at commercial mid-rate
C. leucostoma growth in chemically amended plates
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Several Effective Conventional + Organic ChemicalsIn vitro chemical plates
f f f f f f f f f ff f
e
c
d
b
a a a
0
1000
2000
3000
4000
5000
6000
Mic
roth
iol D
.
Fontelli
s
Torin
o
Pristin
e
Aliette
Topsi
n
Benla
te
Capta
n
Insp
ire S
uperCaC
l
Neem
Oil
Mped
e
Kalig
reen
Seren
ade
NuCop
Badge
ZnSO4
Lime
Sulfur
Positiv
e Contro
l
Chemical Treatments
Les
ion
Are
a (m
m²)
(α = 0.05) Tukey’s HSD adjusted p-values: P < 0.05
Colo
ny A
rea
(mm
2 ) Day 7 Colony Area
Conventional
Organic
Positive Control
Chemical Treatments
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2. Cytospora inoculated onto wounded branches
3. Lesion lengths assessed 8 days post inoculation
Methods Detached branches, chemically submerged
1. Detached branches wounded then submerged in mid-rate chemical solutions for 5 minutes
Conventional chemicals: Aliette, Topsin, Benlate, Captan, Inspire, Ziram
Organic chemicals: Neem oil, Mpede, Kaligreen, Serenade, NuCop, Badge, ZnSO4, lime sulfur
Inoculated detached branches
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C. leucostoma lesion C. leucostoma lesion
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Several Conventional + Organic Chemicals Effective
ConventionalOrganicEffective
a
dd
d
ab
bc
aab
abab
cd
a
a
cd
a
d
0
25
50
75
Aliette
Topsin
Benlat
e
Captan
Inspire
Ziram
Neem O
il
Mpede
Kaligree
n
Serenad
e
NuCopBad
ge
ZnSO4
Lime S
ulfur
Positive
Control
Negati
ve Contro
l
Chemical Treatments
Lesi
on L
engt
h (m
m)
(α = 0.05) Tukey’s HSD adjusted p-values: P < 0.05
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1. Wounds made on 1-year wood2. Label mid-rate chemical sprays
applied after wounding• Conventional: Topsin and
Captan• Organic: Lime Sulfur and
NuCop
4. Branches harvested
MethodsChemical field sprays
3. Inoculation (Summer, Fall, Spring)
Cytospora Inoculation
Cytospora lesion
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Prune wound on 1 year old peach shoot
Chemical applications
Inoculation on peach shoot pruning cut
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Prune
Opening Cytospora infected branch
Cytospora lesion in prune wound
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Topsin and Captan Seasonal Efficacy
Summer Fall Spring
ConventionalOrganicEffective
(α = 0.05) (Tukey’s HSD adjusted p-values: P < 0.05)
2 MPI 5 MPI 3 MPI
Months Post Inoculation (MPI)
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ConventionalOrganicEffective
(α = 0.05) (Tukey’s HSD adjusted p-values: P < 0.05)
Summer Fall Spring
Months Post Inoculation (MPI)
5 MPI 3 MPI
NuCop Potential Phytotoxicity to Peach
2 MPI
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1. Wounds made on 1-year wood
2. Label mid-rate chemical sprays applied after wounding• Conventional: Latex, Topsin and Captan• Organic: kaolin clay (Surround), Lime Sulfur
and NuCop
4. Branches harvested
MethodsChemical paint field sprays
3. Inoculation (Summer, Fall, Spring)
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(α = 0.05) (Tukey’s HSD adjusted p-values: P < 0.05)
Summer Fall Spring
Latex Combinations and Lime Sulfur Show Evidence of Seasonal Efficacy
2 MPI 5 MPI 3 MPI
ConventionalOrganicEffective
Months Post Inoculation (MPI)
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Most Effective Preventive Treatment?
vConventional treatments:vTopsinvCaptanv50% Latex
(Combinations)
vOrganic treatments:vLime Sulfur
Infected Prune Wound on Peach Tree
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Chemical Testing PhasesLaboratory Assay:Testing chemicals in vitro
Laboratory Assay:Testing chemicals on detached branches
Field Trials:• Testing chemical sprays as preventive measure on
branches• Testing chemicals embedded in paints as
preventive measure on branches• Testing chemicals on existing cankers
for spore suppression
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Methods Painting existing cankers1. Cytospora cankers selected randomly in field
2. 10 ml of water (spore effluent) collected from cankers for pre-treatment measurements
3. Chemicals paint combinations applied to cankers:
• Conventional Chemicals: Topsin + latex, Captan + latex, and latex
• Organic Chemicals: Lime sulfur + Surround, NuCop + Surround and Surround
4. Spore effluent collected from cankers after chemical applications once a month for 7 months
Spore effluent collection from canker
Spore counts in hemacytometer
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Pre-treatment Cytospora canker Latex treated Cytospora canker
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(α = 0.05) (CI = 95%) (Tukey adjustment)
Oct. (Pre-trt)Nov.Dec.Jan.Feb.Mar.Apr.May
***
*
* **
*
*
**
**
**
* *
*
*
*
*
Latex Control
Latex + Topsin
Latex + Captan
SurroundControl
Surround +NuCop
Surround +Lime Sulfur
Positive Control
30,000
20,000
10,000
0
= Significant spore decrease compared to pre-treatment (red) within treatment group
Continuous Spore Production in all Treatments
Spor
es C
ount
s (pe
r ml)
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• How many Cytospora species cause cankers and gummosis ?
• What chemicals are effective against Cytospora?– Preventative vs. suppressive?
• When are trees susceptible?
Questions
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0
100
200
300
400
500
600
June Wk 1
Ju ly Wk 1
Ju ly Wk 3
Augus t Wk1
Augus t Wk 3
S ep t Wk 1
S ep t Wk 3
Oc t Wk 1
Oc t Wk 3
Nov Wk 1
Nov Wk 3
De c Wk 1
De c Wk 3
Ja n Wk 1
Ja n Wk 3
F eb Wk 1
F eb Wk 3
Ma rch W
k 1
Ma rch W
k 3
April Wk 1
April Wk 3
Ma y Wk 1
Ma y Wk 3
Lesio
n Vo
lum
e (m
m3 )
Inoculation Date
Lesion Size by Inoculation Month
Cytospora can infect peach at any season
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Cytospora research summary 2016-2017
• Cytospora leucostoma was identified as fungal pathogen
• Chemical options for preventive and suppressive control
• Trees are susceptible year round• The development of a molecular tool to study
epidemiology • Spores are dissemination through the year,
when conditions are favorable
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Treatmentname
Active ingredientLabel rate (per 200
gal.)Rate
chosenMode of Action
MicrothiolDisperss Sulfur 10-20 lb 15 lb Multi-site
Fontelis Penthiopyrad 14-20 oz 17 oz RespirationTorino Cyflufenamid 3.4 oz 3.4 oz UnknownPristine Pyraclostrobin & Boscalid 10.5-14.5 oz 12 oz RespirationAlietteWDG Fosetyl 10 lb 10 lb Unknown
Topsin M WSB Thiopthanate-methyl 1-1.5 lb 1.25 lb Cytoskeleton/
motor proteins
Benlate WP Benomyl 24-32 oz 28 oz Cytoskeleton/ motor proteins
Captan N-Trichloromethylthio-4-cyclohexene-1,2-dicarboximide 3-4 qt 3.5 qt Multi-site
Inspire Super
Difencoconazole &Cyprodinil 16-20 oz 18 oz Protein
synthesis
Ziram Zinc dimethyldithiocarbamate 3 - 5.3 lb 1.15 lb Multi-site
Most Effective Preventive Treatments (Conventional)
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Treatmentname
Active ingredientLabel rate (per 200
gal.)Rate
chosenMode of Action
CaCl CaCl 48 oz 48 oz Multi-siteNeem Oil Neem Oil 3 qt 3 qt Not classifiedMpede Potassium salts 2-4 gal 3 gal. Multi-siteKaligreen Potassium bicarbonate 2.5-3 lb 2.75 lb Not classified
Serenade Bacillus subtilis 14-20 oz 17 ozLipid synthesis/ transport
NuCop WP Copper Hydroxide 8-20 lb 10 lb Multi-site
Badge X2 Copper Hydroxide &Copper Oxychloride 3.5-5.25 lb 4.25 lb Multi-site
ZnSO4 ZnSO4 4-6 lb 5 lb Multi-siteLime sulfur Calcium polysulfide* 20-24 gal. 22 gal. Multi-site
Most Effective Preventive Treatments (Organic)
* Rate was 3% Lime sulfur
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Funding: USDA-CDA Specialty Crop February 2018 – November 2019
Project title: Cytospora management in peach orchards through cultural practices, cultivar selection, and stress mitigation• PIs: Jane E. Stewart & Ioannis S. Minas– Research Associate: David Sterle– Student: Stephan Miller – PhD Research
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Project 1 - Preventive Chemical Applications
• Topsin• Vitaseal• Vitaseal + Topsin• 70% Latex• 50% Latex + Topsin• 70% Latex + Topsin
• JMS Oil + Lime Sulfur• Nufilm + Lime Sulfur• 70% Latex + Lime Sulfur
Started March 20
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Project 2 - Preventive Chemical Applications
Vitaseal + TopsinLatex + TopsinVitaseal + Lime Sulfur
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Glohaven (MI)Glowingstar (MI)Blushingstar (MI)Starfire (MI)Newhaven (MI)Flamin Fury PF19-007 (MI)Flamin Fury PF 23 (MI)Flamin Fury PF 24 (MI)Red Haven (MI)O'Henry (CA)Angelus (CA)Suncrest (CA)
Project 3 - Tolerance of cultivars to Cytospora
Tolerance under high pH and drought
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Dispersal of spores/epidemiologyWind, insects, pruning tools
• Project title: Determining dispersal pathways of Cytospora for the development of management strategies for Cytospora canker on peaches
• PI: Jane E. Stewart– Student: Stephan Miller – PhD Research
Funding: USDA-CDA Specialty Crop February 2019 – November 2020
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Is inoculum spread similar in Colorado?
What are ways spores travel long distances?Wind, insects, humans?
Pattern of disease if spread only by rain splash
Patterns we observe in orchards in Colorado
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The need for a molecular tool for Cytospora identification
• Spores are small and difficult to differentiate
• Cytospora is a slow grower and is out competed by other fungi
Spores of Cytospora leucostoma at 40X
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Marker development as an epidemiology tool
• Genomes of several Colorado isolates were used to identify unique regions in C. leucostoma
• A digital drop molecular assay was developed and tested against closely-related species of Cytospora
Jorge Ibarra-Cabarello
Ibarra-Cabarello et al. Plant Disease. In prep
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Molecular assay identifies only C. leucostoma
• Collect insects and test for vectors
• Test pruning shears
• Test nursery stock
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Thanks!
Collaborators:Greg Litus, Frank Stonaker, Harold Larsen, Jordge Lafantasie, Brady Shanahan, Anne Hess,Bruce Talbott, Cytospora working group – Larry Traubel and Steve Ela
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Cytospora Working Group
Objectives• Collaborate with local commercial growers to
prioritize research efforts• Prevention/Protection measures• Disease management/spread measures• Support in funding opportunities
Want to join? Contact:Jane Stewart: [email protected] Minas: [email protected]
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Species differ in virulence
0
10
20
30
40
50
60
70
80
90
7 14 21 28 35 42 49 57
Candor/C. leucostoma
Sunhaven/C. leucostoma
Sunhaven/C. cincta
Cantor/C. cincta
Time post inoculation (days)
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Biggs. 1986. Phytopathology
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Wang, Iezzoni, Adams Phytopathology.1998.
Species differ in location on tree
Species differ in aggressive on cultivars
C. leucostoma C. cincta C. paraleucostoma
C. leucostoma C. cincta C. paraleucostoma
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Recent Survey of Colorado Orchards
• Estimate incidence and severity of Cytospora in major peach production areas of Western Colorado
• March/April 2015
• Conducted in Grand Valley, North Fork and Olathe areas– Focus on gathering data from
a widespread area and range of orchard management
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Methods• Surveyed by orchard and variety• Recorded presence/absence for every 10th
tree• Counted and rated infection severity for
every 50th tree• Interviewed growers– Irrigation– Orchard floor management– Pruning practices– Pre-plant practices (replant,
fallow, rotation)– Frost protection– Fertilization practices– Pesticide application
K. K im brough