Control of Fungal Pathogens

Megan DewdneyPLP 5115c

Control Measures

oCultural controlsoFungal diseasesoFungicidesoFungicide resistanceoSpecific diseases

Introduction

o Best approach to disease control is to integrate approachesNo one control measure is 100% effective

o Practices such as crop rotation, planting time, (not so easy for perennial crops), site selection, modification of fertilizer regimes, micro-climate modification and sanitation (cultural controls)

o Pesticides

CULTURAL CONTROL

Site and Cultivar/Rootstock Selection

oSite historyWhat was there in the past; was there a disease or nematode

problem?oSoil typepH; drainage; water table

oWhat cultivar do I plan to grow?Highly susceptible to Alternaria; perhaps change planting

distance; less vigorous rootstock

Nursery TreesoKeeping trees clean important as grove

foundationoCovered nurseries started in Brazil to

prevent Citrus Variegated Chlorosis spread

oNow law in Florida and Brazil to prevent spread of HLB

oImportant also to have reliable sources of budwood and rootstocks

Nursery Trees cont.oKeep trees off groundoClean potting mediaoWhat other diseases are controlled in

covered nurseries? Why?

oAlternaria brown spot, citrus scab, citrus canker, black spot, and pseudocercosporafruit and leaf spot

Poor Quality Nursery Trees

Poor Quality Nursery Trees

Poor Quality Nursery Trees

Preventable Disease Problems

Other Cultural Controls

oNO OVERHEAD IRRIGATIONoLeaf litter management is helpful for which diseases? Greasy spot; black spot; alternaria brown spot

oHow?Discing; frequent irrigation to promote decomposition; mulch leaf

litter; put compost accelerator on the leaf litter

More Cultural Controls

oReduce vigorous flush with fertilizer and water managementoIncrease airflow within groveoRemove dead wood and brush pilesoAdjust hedging practicesoRemove declining treesoYounger groves for fresh production

CHEMICAL CONTROL

General Fungicide Information

oOrganic versus inorganicRefers to chemical structure

oInoganic chemicalsSulfur or metal ionsEg. Copper, tin, arsenic, mercury or cadmium

Contact (protectants)

oRemain on surface of plantoPotentially phytotoxicoExcellent for preventative useoPotential problems?Needs to be on the surface before an infection period leads to

repeated applicationsWash off – rain or irrigationUV degradation

Systemics (eradicants, penetrants)oAbsorbed into plant oLocally systemic or translaminarAcross a leaf

oWeakly systemicIntermediate distance

oXylem mobile unidirectional –which way?

oPhloem mobile - bidirectional – why?

Spectrum of Activity

oMulti-siteOften older contact fungicidesUsually effective against many fungal classes

oFew multi-site fungicides being developedRegulatory tests Toxicity (LD50)Environmental regulations (residuals and breakdown products)

Spectrum of Activity

oSingle-siteActive at a sole point in essential processesHighly specific toxicity to fungi therefore generally safe to be

absorbed into plantsOften systemicGreater potential for resistance

Copper

oUsed since before the invention of Bordeaux mixture in 1885 (Millardet)

oBroad spectrum protectantoNon-specificoMOA: Soluble Cu ions (Cu2+) bind tightly to sufhydryl

groupsCystein residues

oPhytotoxicity reduced when made into water insoluble salts

CarbamatesoDiscovered 1930’sFirst group of fully synthetic fungicides

oBroad spectrumoNon-specific inhibitors and are used as protectantsoThe metal salts improve stability oPossible MOA: complex with CuoAscomycetes/Oömycetes

Ethylenebisdithiocarbamate (EBDC)oSecond generation carbamatesGenerally more effective than carbamatesBroad spectrum Non-specific inhibitors and are used as protectantsThe metal salts improve stability

oMOA: slow release of ethylene diisothiocarbamateoAscomycetes/Oömycetes

TrichloromethylthiocarboximidesoDiscovered early 1950’sBroad spectrum Non-specific inhibitors and are used as protectants

oMOA: active group reacts with bases such as thiol groups –forms thiophosgene (highly reactive) Can also be formed during water hydrolysis Can cause phytotoxicity especially with oil

oAscomycetesoNot registered for citrus

Benzimidazoles

oCommercialized in the 1970’sConsidered a breakthroughBroad spectrum Systemic with eradicant activityBenomyl and thiophanate-methyl convert to carbendazime in

aqueous conditions

oAscomycetes and some Basidiomyceteactivity

Benzimidazoles cont.

oMOA: specific binding to fungal β-tubulinPrevents polymerization of microtubulesCells can no longer separate dividing nucleiCan have a cytokinin effect on plants

oResistance!

Sterol Biosynthesis Inhibitors - DMI

oFirst discovered in 1968oLarge class of fungicidesBroad spectrum due to 30 DMI’s availableEach has slightly different properties

oActive against Ascomycetes and BasidiomycetesExcept some Alternaria spp., Colletotrichum spp., Fusarium spp.,

Rhizoctonia spp. or Pyricularia oryzaeReasons unknown

Sterol Biosynthesis Inhibitors – DMI cont.oMost contain an azole group (conazole)oSystemic fungicidesoMOA: The biochemistry is exceptionally well understoodSterols required for membranesFor most fungi it is ergosterolThe aromatic nitrogen group disrupts a cytochrome P450 in

the sterol biosynthe-tic pathway

Sterol Biosynthesis Inhibitors – DMI cont

oOnly essential feature of most DMI’s is the aromatic NoThe sterol precursors build-up and become incorporated into the membranesoNormal membrane functions become disruptedoResistance?

Oömycete Specific FungicidesoPhenylamidesIntroduced in the late 1970’sSystemic with eradicant propertiesStereo-isomers with activity with only the R-isomerMOA: specific for oömycetesInhibits RNA polymerase to that synthesises ribosomal RNAMechanism for oömycete

specificity unknownResistance?

Oömycete Specific Fungicides cont.

oFosetyl-alDiscovered and developed early 1980sSystemic and is translocated to the roots from the leaves (unusual)Has some activity against other pathogens including bacteriaMOA: not understood Possibly acting through phosphonic acid generated in plants or stimulation

of plant defenses Resistance? Starting to hear rumors of resistance

Oömycete Specific Fungicides cont.

oCarboxylic acid amide fungicide (Mandipropamid)Developed in the 2000sProtectant to be applied as drench prior to disease onset

oMOA: Specific for oömycetesActs on cell wall and does not enter the cellInhibits cellulose synthesisResistance?

National Center for Biotechnology Information. PubChem Compound Database; CID=11292824, https://pubchem.ncbi.nlm.nih.gov/compound/11292824 (accessed Sept. 16, 2017).

Strobilurins (Quinone outside inhibitors; QoI)

oIntroduced in the late 1990’soDerived from secondary metabolites of BasidiomyctesoVery broad spectrumAscomycetes, Basidiomycetes and Oömycetes

oSystemic that has mild eradicant abilities

Strobilurins (QoIs) cont.

oMOA: Bind to cytochrome bc1 and inhibit mitochondrial respiration

oPotent inhibitors of spore germination but don’t completely inhibit mycelial growth

oAlternate respirationoResistance?

www.cgm.cnrs-gif.fr/podospora/plus.html

Succinate Dehydrogenase Inhibitors (SDHI)

oDiscovered over 40 years agoFirst generation only had efficacy on BasidiomycetesNew generation with wider efficacy introduced in 2003

oBroad spectrumAscomycetes and Basidiomycetes

oSingle site

https://nl.wikipedia.org/wiki/Boscalid

boscalid

http://www.google.com/patents/EP2612554A1?cl=en

http://www.enlightcorp.com/suppliers/jiangsu_furun_biochemical_technology_co_ltd-hz14fda9e.html

fluxapyroxad

fluopyram

SDHIs cont.

oDivided into 8 chemical classes and 18 compoundsoMOA: Bind to mitochondrial complex II (succinate:quinone

oxidoreductase) and inhibit mitochondrial respirationoPotent inhibitors of spore germination

and mycelial growthoResistance?

Fungicide Resistance

oWhere the target population is no longer sensitive enough to a fungicide to obtain sufficient control

oOccurs in response to the repeated use of a fungicide or related fungicides Field resistanceLaboratory resistance

oOften becomes apparent with sudden total failure of control

Two types of Resistance

Brent and Holloman, 2007

Qualitative

Quantitative

Qualitative Resistance

oSudden and marked loss of controloPresence of two separate populations of resistant and

sensitive pathogen isolatesoUsually a stable form of resistanceLasts for decadesOccasionally will gradually diminish over time but returns rapidly

when fungicide is reapplied without precautions

Quantitative Resistance

oResistance appears less suddenlyDecline in sensitivity of pathogen population and control can be

observedoPopulation tends to revert to sensitive if the fungicide is less

used or is alternated with other MOAs

Types of Resistance

oCross resistanceresistant to different fungicides with the same MOA so generally

same mechanismoMultiple resistanceTwo separate mechanisms of resistance to unrelated MOAs

occasionally occurs in pathogens – multiple resistances

Where does resistance come from?

oA minute proportion of the population carries a mutant form of a gene before the use of a fungicideConfers resistance

oWithout the fungicide, the mutation confers no advantage to growth or survival of the pathogenCould even be deleterious

oMutation could disappear and reappear spontaneously several timesWithin population

Initial frequency 1:1000 millionResistant individual

Increased frequency of resistant strain in population

Several Cycles

Time passes

Resistant population

Resistance cont.

oIf a fungicide is highly effective resistance will be more rapidFewer sensitive strains in population

oIf fungicides are only 80% effective then the buildup of resistance will be slower

Mechanisms of ResistanceoBenzimidazolesThe inhibitor no longer bound to the β-tubulinResulted in rapid and absolute failure

oTo control resistance most Benzimidazoles were mixed with older protectants to control resistant sub-populations

oWas mixed with another β-tubulin inhibitors (phenylcarbamates) but populations resistant to both became apparentUp to 4 separate mutations in the β-tubulin gene

Mechanisms of Resistance

oDemethylation inhibitors (DMIs)oFungal isolates had reduced sensitivity to the fungicidesThe population was still controlled but it was less efficiently

controlledSlow emergence of resistance

oSeveral genes with additive effects but the number appears to be finite

oMechanism is not well understood

Mechanisms of Resistance

oPhenylamideoSimilar situation to the BenzimidazolesComplete fungicide failure

oThe RNA synthase was no longer inhibitedoSome products now have triple mixes of fungicides to try

and control resistant sub-populations

Mechanisms of Resistance

oCarboxylic acid amide fungicideoReported in field isolates of Plasmopara viticolaNone in citrus pathogens to date

oSingle mutation in a gene in the cellulose synthesis pathwayRecessive traitG1105S (amino acid glycine to serine) changes conformation of the

protein and gives resistance

Brum et al., 2010a,b

Mechanisms of Resistance

oStrobilurins (Quinone outside inhibitors; QoI)Resistance occurred within two years of introduction

oSingle mutation in the cytochrome bc1 geneG143A (amino acid glycine to alanine) confers a high level of

resistanceF129L (amino acid phenylalanine to leucine) and G137R (amino

acid glycine to arginine) confer a lower degree of resistance and can be managed by using the recommended rates

What Happens With G143A Type Resistance

oTrees treated with azoxystrobin prior to inoculationSensitive and resistant

isolates of A. alternataBlack bars water treated

control plants

Vega and Dewdney, 2014

Mechanisms of Resistance

oSuccinate Dehydrogenase Inhibitors (SDHIs)Resistance occurs at a regular frequency

oSeveral mutations in the succinate dehydrogenase subunit genesThere are 4 subunits (SDHA, SDHB, SDHC, SDHD) but mutations

have not been found in SDHAA total of 27 resistance conferring mutations have been reported to

date

Mechanisms of Resistance (SDHIs cont.)oIn SDHB: H277Y or H227R (amino acid histidine to tyrosine

or arginine)The histadine is highly conserved in most organisms

oIn SDHC: H134R (amino acid histidine to arginine)Involved with heme b ligation (same function in SDHD)

oIn SDHD: H133R (amino acid histidine to arginine), D123E (amino acid aspartate to glutamic acid)

oModerate resistance conferred by T28A (amino acid threonine to alanine) and A47T in SDHD (amino acid alanine to threonine)

Resistance Management

oDo not use one product exclusivelyUse in rotation or in a mixture with another MOA

oRestrict the number of applications or quantity of AI/area/year

oMaintain the recommended doseoIPMoUse as many AI’s as possibleoSpray when the population is smallest

MANAGEMENT OF DISEASES

Greasy Spot Disease Cycle Caused by Zasmidium citri-griseum

Cultural Controls

oReduce leaf litter in winter and early springDiskingFrequent irrigation to promote decompositionMulch leaf litterPut urea or lime on the leaf litter

oProblem with this approachNot enough of the leaf litter is decomposed

Spray Timing Greasy Spot

oLess susceptible cultivarsOne spray between May and June often sufficient especially in

Northern production regionsoIn South Florida, more susceptible cultivars and in groves

with severe defoliationTwo sprays; one mid-May – June, the second once flush has

expandedoResistance risks?

Fungicides Recommended for Greasy SpotoPetroleum oil – gives adequate control on less susceptible

cultivars (FRAC NC)oCopper – more consistent control than oil for fruit (FRAC M

01)oQuinone oxidase inhibitors (QoI) or strobilurins (FRAC 11)Azoxystrobin; Trifloxystrobin; Pyraclostrobin

oFenbuconazole (FRAC 3)DMI fungicide or sterol biosynthesis inhibitors

oPremixturesAzoxystrobin and defenoconazole (FRAC 11 + 3)Pyraclostrobin and boscalid (FRAC 11 + 7)

Melanose Disease Cycle Caused by Diaporthe citri

Cultural Controls

oSelect younger groves for fresh fruitLess dead wood for inoculum production

oRemove dead wood from canopySmall twigs are easily missed or overlooked

oClear out brush piles

Fungicide Recommendations for MelanoseoCopper –Most economical but can cause blemishes in hot

dry weather (FRAC M 01)oQuinone oxidase inhibitors (QoI) or strobilurins – Low residual

activity compared to copper but useful in hot weather (FRAC 11)AzoxystrobinTrifloxystrobinPyraclostrobin

oPremixtures – contain rotational modes of actionAzoxystrobin and difenoconazole (DMI) (FRAC 11 + 3)Pyraclostrobin and boscalid (SDHI) (FRAC 11 + 7)

Spray TimingoOranges and TangerinesFirst spray mid to late AprilOne to two applications sufficient

oGrapefruit (fresh market)First application when fruit ¼ to ½ inch in diameterCopper to be applied every 3 weeks until fruit resistant in late June

to early JulyThere is a model to determine whether copper residues are

sufficient to control disease based on weathering of copper and the growth rate of fruit

Post Bloom Fruit Drop Disease Cycle Caused by Colletotrichum acutatum

Cultural Controls

oNo overhead irrigationIf necessary only at nightWhat is the rational behind this recommendation

oIf there are trees in decline from other diseases such as tristeza, blight, phytophthora root rot, or HLB that promote off season bloom, remove them from your PFD prone block

Fungicides Recommended for PFDoFerbam (FRAC M 03)oQuinone oxidase inhibitors (QoI) or strobilurins (FRAC 11)AzoxystrobinTrifloxystrobinPyraclostrobin

oPremixed fungicidesAzoxystrobin and difenoconazole (DMI) (FRAC 11 + 3)Pyraclostrobin and boscalid (SDHI) (FRAC 11 + 7)Pyraclostrobin and fluxapyroxad (SDHI) (FRAC 11 + 7)

Fungicides Postbloom Fruit Drop

oPremixed Cont.Pyraclostrobin and fluxapyroxad (SDHI) (FRAC 11 + 7)Trifloxystrobin and fluopyram (SDHI) (FRAC 11 + 7)

oShould there be concerns with choices available and why?

Predict PFD infection periods oCitrus Advisory SystemoHosted on Agroclimate.orgUnder tools/crop diseasesEach circle represents a

FAWN weather stationFlorida Agricultural Weather

Networkhttps://fawn.ifas.ufl.edu/

Black Spot Disease Cycle Caused by Phyllosticta citricarpa

Cultural ControlsoIncrease air flow in trees to reduce leaf wetness where

possibleoAvoid cultivars with significant off-season bloomOlder fruit can supply inoculum to infect new fruit

oReduce leaf litter to reduce inoculumoMinimize trash when picking to avoid inadvertent

movement of the fungus from one location to anotheroClean equipment between groves with disinfestantsQuaternary ammonium (2000 ppm) or bleach (200 ppm)

Fungicide Recommendations for Black SpotoCoppers (FRAC M 01)oQuinone oxidase inhibitors (QoI) or strobilurins (FRAC 11)AzoxystrobinPyraclostrobinTrifloxystrobin

oFenbuconazole (FRAC 3)oPremixed fungicidesAzoxystrobin and difenoconazole (DMI) (FRAC 11 + 3)Pyraclostrobin and boscalid (SDHI) (FRAC 11 + 7)

Good News on the Resistance Front

o Intron following the G143 codonG143A mutation interferes with splicing of condonMutation never described in fungi with similar intron

Described by Stammler et al. 2013 and Hincapie et al., 2014oMutations F129L or G137R can still occur but level of resistance

is much lowerPotential rescue mechanisms proposedIntron deletionOverexpression of genes coding for mitochondria metal ion carriers in nucleusA second mitochondrial mutation to restore exon/intron splicing

Hincapie et al., 2014

Vallières et al., 2011

Black Spot Application Timing

Late Spring (April/May)

Copper

Continue applications at 1 month intervals

oFruit is susceptible for 5-6 months post-petal fallCopper and/or other fungicide

CopperCopper and/or other fungicide

Copper

Use other fungicides when concerned about copper phytotoxicity

Alternaria Brown Spot Disease Cycle Caused by Alternaria alternata

Cultural ControlsoDisease-free nursery treesoCareful choice of planting siteAir drainage importantWider spacing

oNo vigorous rootstocksoNo over-fertilization or over-wateringoHedge in late MarchoNo overhead irrigation

Fungicides Recommended for AlternariaoCopper – Works well for fruit but not leaves (FRAC M 01)oFerbam – Only moderately effective (FRAC M 03)oQuinone oxidase inhibitors (QoI) or strobilurins (FRAC 11)AzoxystrobinTrifloxystrobinPyraclostrobin

oPremixed fungicides – two modes of actionAzoxytrobin and difenoconazole (FRAC 11 + 3)Pyraclostrobin and boscalid (FRAC 11 + 7)

Spray Timing

oFirst spray when spring flush ¼-1/2 full expansionWith high inoculum another before full expansion or at petal fall

oRest of the year maintain protective coatingoAny concerns with fungicide choices?

Citrus Scab/Sweet Orange Scab Disease Cycles Caused by Elsinoë fawcetti and E.australis

Cultural Controls

oDisease-free nursery treesStart clean and problems are unlikely

oHedge and top badly-affected plantingsDoes not move far even within trees

oNo vigorous rootstocksoNo overhead irrigation

Fungicide Recommendations for Scab

oCopper (FRAC M 01)oFerbam (FRAC M 03)oQuinone oxidase inhibitors (QoI) or strobilurins (FRAC 11)AzoxystrobinTrifloxystrobinPyraclostrobin

oFenbuconazole (FRAC 3)

Spray Timing Scab

oSprays are mainly for groves with a recent history of ScaboFirst spray – spring flush 2-3 inches can be omitted if severity was light

oPetal falloThree week after petal fall

Sources

http://apsnet.org/education/IntroPlantPath/Topics/fungicides/default.htm

Köller, W. 1999. Chemical approaches to managing plant pathogens. In Handbook of Pest Management. ed. Ruberson, J.R. Marcel Dekker, New York p. 375.

https://www.frac.info/docs/default-source/publications/frac-code-list/frac-code-list-2019.pdf?sfvrsn=98ff4b9a_2Updated annually

Sources cont.

https://crec.ifas.ufl.edu/resources/production-guide/This guide is updated annually. If citing as a source, use the most

recent version and verify that the management recommendations continue to be the same. – They change with new information.