Chapter 28 Resistance in Postharvest Pathogens of Citrus in the United States

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1 Chapter 28 Resistance in Postharvest Pathogens of Citrus in the United States James E. Adaskaveg and Helga Förster Abstract Among citrus postharvest pathogens, fungicide resistance in the United States to date has only been reported for species of Penicillium. Except for the inorganic salts, widespread resistance has developed to all of the older fungicides such as the still registered phenylphenols o -phenylphenol and sodium o-phenylphenate, the methyl benzimidazole carbamate, thiabendazole, and the demethylation inhibitor imazalil. The almost simultaneous introduction of several new compounds in the early 2000s that include the anilinopyrimidine pyrimethanil, the phenylpyrrole fludioxonil, and the quinone outside inhibitor azoxystrobin offered a unique opportunity in keeping the development of resistance to a minimum. Fungicide modes of action could be mixed and rotated from the first introduction before resistance had occurred. Resistance to pyrimethanil, however, has developed in some packinghouse populations of P. digitatum because end users did not follow these guidelines and used the fungicide exclusively. For azoxystrobin, resistance in P. digitatum has only been described for laboratory mutants and for fludioxonil only in mass platings of conidia on selective media in the laboratory or in packinghouse air samplings. Thus, practical resistance to azoxystrobin and fludioxonil has not occurred. Natural resistance frequencies and molecular mechanisms for thiabendazole, imazalil, pyrimethanil, fludioxonil, and azoxystrobin have been studied, and resistant pathogen isolates have been evaluated for their fitness. Anti-resistance strategies focus on sanitation of fruit, equipment, and storage rooms; limitation of pathogen sporulation and spore dispersal; use of fungicide mixtures, pre-mixtures, and rotations; as well as the early detection of resistance. Keywords Postharvest decays Penicillium spp. Green mold Postharvest fungicides Resistance development Resistance management J.E. Adaskaveg (*) H. Förster Department of Plant Pathology and Microbiology, University of California, Riverside, CA, USA jim.adaskaveg@ucr.edu Springer Japan 2015 H. Ishii, D.W. Hollomon (eds.), Fungicide Resistance in Plant Pathogens, DOI / _28 449

2 450 J.E. Adaskaveg and H. Förster 28.1 Introduction The main citrus-growing areas in the United States are Florida, California, Texas, and Arizona. In 2012, Florida produced 63 % of the total citrus crop with oranges and grapefruit accounting for about 70 % and 65 % of the country s production, respectively ( ). California produced 34 % of the citrus, providing 92 % of the lemon and 80 % of the tangerine crop. With current major disease threats to the Florida industry (i.e., citrus greening Huanglongbing, bacterial canker), production in this state is expected to decline dramatically over the next years. Although California ranks second in production, postharvest fungicide use on citrus surpasses that of Florida. This is because Florida oranges are mainly used for juicing, while most of the California crop is used for fresh fruit consumption and has to arrive decay-free and with high quality to the final consumer. Additionally, California lemon fruit may be stored for up to 3 or 4 months in the packinghouse before marketing based on economic needs and incentives. With extensive handling of fruit during harvest, transport to packinghouse, grading, packing, storage, and final marketing that often includes long-distance transport to worldwide destinations, fruit may become infected with decay organisms at any stage of the processing chain and thus have to be protected from decay development. This is most effectively done in an integrated postharvest decay management program that includes the application of postharvest fungicides (Adaskaveg et al ). Postharvest decay is one of the most important factors diminishing value and limiting marketing of fresh citrus. Losses of fresh citrus by decay after harvest are more costly than those occurring before harvest because of the added expenses for harvesting, postharvest handling, treatments, shipping, and storage. Occurrence and severity of postharvest decays depend on many factors including growing region and environmental conditions, fruit variety, tree condition, cultural practices, preharvest treatments, harvest methods, and postharvest handling practices Major Postharvest Decays of Citrus Green mold, caused by the fungus Penicillium digitatum, is the most important postharvest decay of citrus fruit. It occurs year-round on all citrus varieties worldwide, but especially in locations with arid and subtropical climates (Eckert and Eaks 1989 ). Blue mold, caused by P. italicum, is generally less prevalent, but may become a major problem under conditions that suppress development of green mold such as storage temperatures below 10 C (Brown and Eckert 2000 ). In California, green and blue molds, as well as Alternaria decay, may also be major problems when orange, mandarin, and lemon fruits are de-greened with ethylene immediately after harvest in non-refrigerated storage rooms (e.g., C) without preharvest or postharvest fungicide and sanitation treatments (Eckert and Eaks 1989 ). In other

3 28 Resistance in Postharvest Pathogens of Citrus in the United States 451 regions such as Florida, de-greening results in higher incidence of other decays such as anthracnose caused by Colletotrichum species and stem-end rots (see below). Sour rot, caused by Galactomyces citri-aurantii (anamorph, Geotrichum citriaurantii ), ranks second in postharvest decay losses after Penicillium decays. In California, sour rot is particularly problematic on lemons grown in the coastal regions and on mandarins that are often stored for extended periods at relatively high temperatures of C and high relative humidity (92 98 %) to obtain marketable rind color (Eckert 1959 ; Suprapta et al ). Overall, outbreaks of the disease are sporadic with highest incidences on those fruits that are harvested during prolonged wet conditions (Baudoin and Eckert 1982 ; Eckert 1959 ). In storage, the disease may result in complete collapse and liquefaction of infected fruit. Juices dripping from infected fruit can readily spread the pathogen to healthy fruit (Eckert and Eaks 1989 ). Other postharvest decays of citrus fruits include brown rot (caused by several species of Phytophthora, mostly P. citrophthora, P. syringae, P. parasitica, P. hibernalis, and P. palmivora ), gray mold (mostly on lemons; caused by Botrytis cinerea ), anthracnose (caused by Colletotrichum gloeosporioides ), Septoria spot (caused by Septoria citri ), Alternaria fruit rot (caused by Alternaria alternata ), whisker mold (caused by P. ulaiense ), and Trichoderma rot (caused by Trichoderma viride ) (Timmer et al ). Stem- and blossom-end rots are more prevalent in the humid climate of Florida and may be caused by Alternaria citri, Botryosphaeria rhodina, Phomopsis citri, and other species (Timmer et al ) Fungicides Registered for Postharvest Use on Citrus in the United States and Development of Resistance in Postharvest Pathogen Populations Due to their high incidence and severity, Penicillium decays have been the primary targets in the development of citrus postharvest fungicide treatments. All compounds registered to date have at least some activity against these decays (Table 28.1 ). The recently registered propiconazole is the only fungicide that also has high activity against sour rot. In other parts of the world, however, guazatine is available and is effective against sour rot and Penicillium decays (Rippon and Morris 1981 ). Some of the first treatments used to control postharvest decays of citrus fruits were alkaline solutions of borax, sodium carbonate (soda ash), and sodium bicarbonate. In California, their beneficial action to reduce the incidence of Penicillium molds of citrus was first realized over 80 years ago (Barger 1928 ). In addition to these inorganic salts and the phenylphenols, citrus postharvest fungicides represent six FRAC (Fungicide Resistance Action Committee; ) groups that all target a single-site mode of action: the methyl benzimidazole carbamates (MBCs; FRAC 1), the sterol demethylation inhibitors (DMIs; FRAC 3), the anilinopyrimidines (FRAC 9), the quinone outside inhibitors

4 452 J.E. Adaskaveg and H. Förster Table 28.1 Fungicides registered in the United States as postharvest treatments on citrus to prevent decays caused by fungi Fungicide class Chemicals and trade name Inorganic salt Sodium borate (borax sodium tetraborate), sodium carbonate (soda ash), sodium bicarbonate (baking soda) Phenol derivative Phenylphenols: o-phenylphenol (OPP), sodium o-phenylphenate (tetrahydrate) (SOPP) Methyl benzimidazole 2-(4-Thiazolyl) carbamate (MBC) benzimidazole (thiabendazole TBZ, Mertect 340, Alumni, Decco Salt No. 19) Sterol demethylation inhibitor (DMI) triazole Sterol demethylation inhibitor (DMI) imidazole FRAC group Main targets of activity Penicillium spp. Geotrichum and Penicillium spp., stem-end rots, Trichoderma spp. 1 Penicillium spp., stem-end rots Propiconazole (Mentor) 3 Penicillium spp., Geotrichum citri-aurantii Imazalil (Deccocil, 3 Penicillium spp. Fungaflor, Freshgard, and others) Anilinopyrimidine Pyrimethanil (Penbotec) 9 Penicillium spp. Quinone outside inhibitor Azoxystrobin (Diploma)a 11 Penicillium spp. (QoI, strobilurin) Phenylpyrrole Fludioxonil (Graduate) 12 Penicillium and Botrytis spp., stem-end rots Phosphonate Phosphorous acid (potassium and calcium phosphite Fungi-Phite, ProPhyt, and 33 Mostly Phytophthora spp., some activity against Penicillium spp. others) MBC + phenylpyrrole TBZ + fludioxonil (Graduate Max) Penicillium and Botrytis spp., stem-end rots DMI + anilinopyrimidine Imazalil + pyrimethanil (Philabuster) QoI + phenylpyrrole Azoxystrobin + fludioxonil (Graduate A+) a Only sold in a pre-mixture at this time (i.e., Graduate A+) Penicillium and Botrytis spp., stem-end rots, anthracnose Penicillium and Botrytis spp., stem-end rots (QoIs, strobilurins; FRAC 11), the phenylpyrroles (FRAC 12), and the phosphonates (FRAC 33) (Adaskaveg et al ; Adaskaveg and Förster 2010 ). Resistance in Penicillium populations has developed in selected populations to all of the older fungicides, except for the inorganic salts (Table 28.2 ). The phenylphenols, thiabendazole, and imazalil each were introduced years apart and were then often used exclusively after resistance to the previously registered compounds had already occurred: o -phenylphenol in the 1930s, thiabendazole in the 1970s, and

5 28 Resistance in Postharvest Pathogens of Citrus in the United States 453 Table 28.2 Resistance development in decay organisms of citrus against postharvest fungicides in the United States Fungicide class FRAC Year introduced group Fungicide on citrus Currently registered First occurrence of postharvest resistance Year Decay organism Reference Phenol derivative OPP, SOPP 1936 Yes 1959 Penicillium digitatum Harding (1962 ) Aromatic hydrocarbon 14 Biphenyl 1944 No 1962 Penicillium spp. Harding (1962 ) Aliphatic amine 2-Aminobutane 1962 No 1976 Penicillium spp. Harding (1976 ) Methyl benzimidazole carbamate (MBC) Sterol demethylation inhibitor (DMI) imidazole 1 Benomyl 1967 No 1973 P. digitatum Wild (1983) Thiabendazole 1967 Yes 1970 Penicillium spp. Harding (1972 ) and Muirhead ( 1974 ) 3 Imazalil 1980 Yes 1982 (lab, subsequently in packinghouses) 1987 (lab, subsequently in packinghouses) P. italicum De Waard et al. ( 1982 ) P. digitatum Eckert (1987 ) DMI triazole 3 Propiconazole 2008 Yes Present at introductiona P. digitatum McKay et al. ( 2012 ) Anilinopyrimidine 9 Pyrimethanil 2005 Yes 2009 P. digitatum Kanetis et al. ( 2010 ) Quinone outside inhibitor 11 Azoxystrobin 2008 Yes 2011 (lab)b P. digitatum Zhang et al. ( 2009 ) (QoI, strobilurin) Phenylpyrrole 12 Fludioxonil 2006 Yes 2010 (packinghouse air samplings) b P. digitatum Kanetis et al. ( 2010 ) 2012 c P. italicum Adaskaveg unpublished a Cross-resistance to imazalil b Resistance only reported from laboratory mutants or in packinghouse air samplings, no practical resistance to date c Resistance found at a single location, no practical resistance to date

6 454 J.E. Adaskaveg and H. Förster Fig Lemon fruit inoculated with isolates of Penicillium digitatum sensitive ( a, b ) or resistant ( c, d ) to pyrimethanil and not treated ( a, c ) or treated with 1000 mg/l pyrimethanil ( b, d ) imazalil in the 1980s. The phenylphenols are no longer widely used because of costly disposal of re-collected, spent material. Currently, resistance to thiabendazole and imazalil is very common. The molecular mechanism for MBC resistance in P. digitatum was determined to be a point mutation at codon 198 of the β-tubulin gene where glutamine was replaced with lysine (Koenraadt et al ) or at codon 200 where thymine was replaced by adenine (Schmidt et al ). For the DMI imazalil, several molecular mechanisms have been characterized in P. digitatum. These include a tandem repeat of a transcriptional enhancer in the promoter region of CYP51 that leads to overexpression of the gene (Hamamoto et al ) and a unique 199-bp insert within the transcriptional enhancer unit (Ghosoph et al ). In contrast to the older compounds, the newer fungicides pyrimethanil, fludioxonil, and azoxystrobin were introduced almost simultaneously. This offered a unique opportunity in keeping the development of resistant pathogen populations to a minimum because fungicide modes of action (FRAC groups) could be mixed and rotated. Still, some packers chose to exclusively use a single fungicide based solely on economic reasons or, simply, use the least costly treatment. This resulted in the development of pyrimethanil resistance in populations of P. digitatum, rendering this treatment ineffective in some packinghouses in California (Adaskaveg, unpublished ) (Fig ). Pyrimethanil-resistant isolates of the pathogen were also readily obtained in mass platings of conidia on selective media in the laboratory or

7 28 Resistance in Postharvest Pathogens of Citrus in the United States 455 in packinghouse air samplings, and EC 50 values for mycelial growth were >8 μg/ ml (Kanetis et al ). Anilinopyrimidines are classified as amino-acid and protein synthesis inhibitors by FRAC ( FRAC ), and biosynthesis of methionine and other amino acids was originally described as the mode of action for pyrimethanil in B. cinerea (Masner et al ). This was questioned in subsequent investigations (Leroux et al ; Fritz et al ; Kanetis et al. 2008b ). In studies with P. digitatum, growth of sensitive isolates was inhibited by pyrimethanil even in the presence of methionine, indicating a mode of action different from methionine biosynthesis also in this pathogen (Kanetis et al. 2008b ). Isolates of P. digitatum resistant to fludioxonil, which targets the mitogenactivated protein kinase pathway in this organism (Kanetis et al. 2008b ), have only been obtained in mass selections of conidia in the laboratory or from packinghouse air samplings (Kanetis et al ). Isolates obtained from selection plates could be placed into two categories: moderately resistant isolates with EC 50 values 1 μg/ml and highly resistant isolates with EC 50 values >1 μg/ml (Fig ). All were pathogenic in fruit inoculation studies although less virulent than the wild type. Treatments with fludioxonil were not effective or were reduced in their efficacy when fruits were inoculated with resistant isolates as compared to the wild-type sensitive isolate. To date, however, no fludioxonil-resistant isolates of P. digitatum have been obtained from decayed fruit in packinghouses. A first detection of fludioxonil resistance in P. italicum from treated lemon fruit, however, occurred in 2012 in California with measured EC 50 values greater than 10 μg/ml (Adaskaveg unpublished ). Resistance in P. digitatum to azoxystrobin currently has only been reported from UV-induced laboratory mutants and the molecular mechanism was identified as a G143A mutation in the cytochrome b gene (Zhang et al ). This pathogen was rated as likely to develop high levels of azoxystrobin resistance based on the genetic stability of resistant mutants and absence of a type I intron in the cytochrome b gene directly after codon 143 which has been correlated with a high potential for QoI resistance (Sierotzki et al ; Zhang et al ). Still, despite extensive surveys, no resistance to azoxystrobin was found in Penicillium spp. in citrus packinghouse air samplings or in isolates from treated, decayed fruit (Kanetis et al ). More recently, however, some isolates of P. digitatum from decayed fruit in packinghouse monitoring surveys showed a five- to tenfold reduction in sensitivity against this fungicide (Adaskaveg unpublished ). Thus, among citrus postharvest pathogens, fungicide resistance has only been reported for species of Penicillium. No resistance to the DMI propiconazole has been observed in G. citri-aurantii although this pathogen has been exposed to the DMI imazalil for many years (McKay et al ). Additionally, in mass platings of conidia and in soil population enrichment assays with G. citri-aurantii, no isolates with reduced sensitivity could be recovered. This is in contrast to experiments with propiconazole-sensitive isolates of P. digitatum where resistant isolates were readily obtained in platings of conidia from propiconazole-sensitive isolates (McKay et al ). Propiconazole-resistant isolates of Penicillium spp., however, commonly occur due to cross-resistance with imazalil. Table 28.3 summarizes the current

8 456 J.E. Adaskaveg and H. Förster Fig Lemon fruit inoculated with isolates of Penicillium digitatum sensitive ( a, b ), moderately resistant ( c, d ), or highly resistant ( e, f ) to fludioxonil and not treated ( a, c, e ) or treated with 1000 mg/l fludioxonil ( b, d, f )

9 28 Resistance in Postharvest Pathogens of Citrus in the United States 457 Table 28.3 Resistance potential to four citrus postharvest fungicides in populations of P. digitatum and characteristics of resistant isolates a Characteristic Azoxystrobin Fludioxonil Pyrimethanil Propiconazole Laboratory selection of resistant No Yes Yes Yes isolates Packinghouse selection of resistant No Yes Yes Yes isolates Practical resistance (packinghouse) No No Yes (locally) Yes Natural resistance frequency Resistance factor MR: 3 26; HR: >1,500 b > Pathogenicity of resistant isolates Similar to wild type, sometimes Similar to wild type Similar to wild type reduced Fitness of resistant isolates HR: not fit; MR: reduced fitness as compared to wild type a Data from Kanetis et al. ( 2010 ), Förster and Adaskaveg ( 2012 ), and McKay et al. ( 2012 ) b MR moderately resistant isolates, HR highly resistant isolates Similar to wild type Not determined

10 458 J.E. Adaskaveg and H. Förster occurrence of resistance in P. digitatum to the newer fungicides azoxystrobin, fludioxonil, pyrimethanil, and propiconazole and characteristics of resistant isolates that are further discussed below. Multiple resistance (resistance to different classes of fungicides that have unique modes of action) has also been found in P. digitatum populations. For example, resistance was present to biphenyl and SOPP (Harding 1962 ) or to 2-aminobutane, SOPP, TBZ, and benomyl (Davé et al ). In another report, multiple resistance developed in populations of Penicillium spp. from citrus where thiabendazole and imazalil were introduced after resistance had already developed against the previously registered biphenyl and the phenylphenols (Holmes and Eckert 1999 ) Factors Contributing to Resistance Development in Postharvest Decay Pathogens of Citrus Resistant pathogen populations develop gradually by selection and subsequent proliferation of rare naturally occurring individuals in the population that are less sensitive to a specific chemical (Brent and Hollomon 1998 ). The frequency of the less sensitive individuals in the population (i.e., the natural resistance frequency) and the risk for resistance development are determined by the pathogen and the fungicide s mode of action. With their enormous asexual reproduction potential, Penicillium species are ranked at high risk for resistance development (Brent and Hollomon 1998 ). Resistance frequencies have been quantified for some pathogenfungicide combinations. Thus, the resistance frequency for the MBC benomyl in unselected orchard populations of P. digitatum was determined at approximately 10 7 to 10 8 (Wild 1980 ; Eckert 1988 ). In mass platings of conidia of this pathogen, ranges of resistance frequencies in laboratory and packinghouse populations for the newer postharvest fungicides fludioxonil, pyrimethanil, and propiconazole were to , to , and to , respectively (Kanetis et al ; McKay et al ; Table 28.3 ). In contrast, similar tests conducted with G. citri-aurantii did not yield any resistant individuals, indicating that perhaps this fungus is less prone to resistance development (McKay et al ). All of the newer postharvest fungicides are single-site mode of action compounds, and these intrinsically have a much higher risk for resistance development than multisite mode of action compounds (Hewitt 1998 ; Kendall and Hollomon 1998 ). The speed of resistance development after the initial selection depends largely on the presence of continued selection pressure (e.g., stability of fungicide residue on treated fruit during storage, repeated applications of the same fungicide class to the same fruit lot) and the competitive fitness of the less sensitive isolates. Resistant pathogen isolates have been evaluated for their fitness based on pathogenicity (ability to cause decay), decay rate (growth rate of pathogen), virulence (severity of decay), and sporulation potential (reproductive ability). For the qualitative, single-step type of resistance that is the result of a mutation in a single or, at most,

11 28 Resistance in Postharvest Pathogens of Citrus in the United States 459 a small number of major genes (Kendall and Hollomon 1998 ) and that is typical for the MBC fungicides, resistant populations generally remain stable in the absence of selection pressure (Brent and Hollomon 2007 ). Thus, there are limited fitness penalties in isolates with qualitative resistance, but exceptions are known such as with phenylamide resistance in Phytophthora infestans (Cooke et al ). Isolates of P. digitatum resistant to MBC compounds, however, showed a somewhat reduced virulence, and the resistant biotype decreased in frequency in the absence of selection pressure in laboratory competition studies as well as in packinghouses (Wild 1980 ; Eckert 1988 ). In contrast, current packinghouse populations of P. digitatum with thiabendazole resistance (EC 50 values >7.8 μg/ml) have been found to be stable and persistent (Adaskaveg 2004 ). Fitness penalties commonly occur in pathogens with quantitative, multistep resistance that is the result of mutation of several genes and that is typical for DMI fungicides. This type of resistance rapidly reverts to a more sensitive condition in the absence of selection pressure ( Brent 1995). In agar and fruit assays with P. digitatum, the frequency of resistant isolates declined over two disease cycles when agar plates or lemon fruits were inoculated with mixtures of imazalil-sensitive and imazalil-resistant strains (Holmes and Eckert 1995 ). This reduced fitness of resistant isolates results in an annual fluctuation of imazalil resistance in populations of P. digitatum in citrus packinghouses. Thus, there is a gradual increase in imazalil resistance over the year, with the lowest incidence of resistance during the off-peak season in late fall and early winter and the highest incidence during the main harvest and storage periods in late winter and spring (Adaskaveg 2004 ). Fitness and competitiveness of resistant isolates of P. digitatum have also been evaluated for the newer fungicides fludioxonil and pyrimethanil. Pyrimethanilresistant isolates with EC 50 values of 2 to >10 mg/l (baseline, <0.7 mg/l) and fludioxonil-resistant isolates with EC 50 values of 0.1 to >10 mg/l (baseline, <0.02 mg/l) were pathogenic on citrus fruit. Isolates resistant to pyrimethanil were mostly similar in growth, virulence, and sporulation to wild-type sensitive isolates (Fig ), whereas isolates resistant to fludioxonil displayed a range of growth, virulence levels, and sporulation capacities (Fig ). Resistant isolates were sometimes less virulent and most isolates showed reduced sporulation as compared to wild-type sensitive isolates (Kanetis et al ). Isolates highly resistant to fludioxonil were not recovered from decaying fruit after co-inoculation with sensitive isolates; recovery rates for isolates moderately resistant to this fungicide were % (Förster and Adaskaveg 2012 ). Isolates highly resistant to pyrimethanil, in contrast, were recovered at frequencies between 14.8 and 84.1 %. Thus, these studies demonstrated fitness differences for different modes of action among resistant isolates of P. digitatum. Fitness penalties exist for fludioxonil resistance but are less apparent for pyrimethanil resistance indicating that without proper anti-resistance strategies, resistant isolates may become predominant after repeated fungicide applications. Isolates from fludioxonil and pyrimethanil selection plates were also stable in their fungicide sensitivity characteristics after several passages on nonamended agar and disease could not be sufficiently controlled after treatment with the respective fungicides (Kanetis et al ).

12 460 J.E. Adaskaveg and H. Förster Postharvest handling practices can also affect the risk of resistance development. As indicated above, some California citrus fruits, especially lemons, may be stored for long time periods in the packinghouse before marketing. Decay may develop in storage, leading to an initial selection process on the fungicide-treated fruit. When fruits are taken out of storage before shipment to market, they are repacked, and airborne inoculum from decayed fruit is readily dispersed to healthy fruit or to fruit coming from the field if strict sanitation practices are not followed which include the spatial separation of these different packinghouse activities. This inoculation potential is exacerbated when fruits were treated with a postharvest fungicide that has no antisporulation activity such as pyrimethanil. Fruits may then receive a second fungicide application, sometimes with the same fungicide that was used before storage. In California lemon production, the risk of fungicide resistance development in Penicillium populations is further increased by the year-round availability of susceptible fruit in the packinghouse because fruits are harvested, processed, and stored almost continuously throughout the year without a break (Adaskaveg et al ) Resistance Management The overall goal of fungicide resistance management is to achieve sustainable, highly effective disease management with the use of fungicides now and in the future. Specific objectives are to minimize the development of new resistance in pathogen populations (i.e., to fungicides against which resistance was never reported previously and in new fungal species fungicide combinations), to detect the development of resistance at an early stage, and to mitigate the impact of resistance in managing diseases so that crops can be economically produced and distributed to the consumer. To achieve these goals, decay management programs generally have to be modified at several levels. Resistance has not developed to date in commercial situations in the United States against several of the newer postharvest fungicides (i.e., azoxystrobin and fludioxonil in Penicillium species, propiconazole in G. citriaurantii ) and the challenge is to protect their efficacy. Table 28.4 summarizes postharvest practices that help mitigating the development of resistance. Below we discuss some of those practices that are directly related to fungicide use and reduction of pathogen populations (e.g., sanitation, anti-sporulation methods). Information on other practices that are beyond the scope of this chapter can be found in several detailed publications (see Kader 2002 ). Early detection of resistance and of the composition of the pathogen population allows the targeted adjustment of fungicide usage. This is done by implementing routine monitoring programs where fungicide sensitivities of the current pathogen population are compared to those of a population that was sampled before the use of a new fungicide (baseline sensitivity). Baseline sensitivities for isolates of P. digitatum and P. italicum in California have been established for azoxystrobin, fludioxonil, and pyrimethanil (Kanetis et al. 2008a ) and for G. citri-aurantii for propiconazole (McKay et al ). Additionally, new high-throughput monitoring methods have

13 28 Resistance in Postharvest Pathogens of Citrus in the United States 461 Table 28.4 Resistance management strategies used in a packinghouse Overall strategy Specific practice Benefit Optimize health of the fruit Use sanitizing methods (chemical and physical) Optimize fungicide use Optimize fungicide efficacy Early detection of resistance Minimize injuries during harvest and handling Keep fruit at optimal temperature during storage, transportation, and marketing Limit storage times Sanitize healthy and decayed fruit, handling equipment, and storage rooms Filter air to reduce spore load and regulate air flow Remove cull fruit from packinghouse Spatially separate incoming fruit from the field from the repacking area in the packinghouse Use rotations and mixtures or pre-mixtures whenever possible before resistance selection occurs Limit the total number of fungicide applications of any one class ideally to one per fruit lot Use labeled rates Use fungicides that inhibit sporulation on fruit that will be stored Use optimal application methods (e.g., high volume drenches are better than low-volume sprays) Use heated (50 55 C) fungicide solutions Integrate use of alkaline salts with fungicide programs Use compatible sanitizers in recirculating fungicide solutions Use fungicide mixtures, premixtures, and rotations Routinely monitor pathogen populations on decayed fruit for fungicide sensitivity Most postharvest pathogens are wound pathogens and minimizing injuries reduces decay Senescence is delayed and fruits are less susceptible to decay Senescence is reduced and fruits are less susceptible to decay. The chance of decay development is reduced Pathogen populations are reduced and contamination of healthy fruit is minimized Inoculum dispersal among different sections of the packinghouse is reduced Resistance that was potentially selected on treated fruit is moved away from packinghouse Prevent introduction of new pathogens or pathogens that have been selected for resistance Resistance risk is reduced when pathogens are exposed to more than one fungicide mode of action Resistance risk is reduced in single exposures Sublethal rates may select for less sensitive pathogen individuals Selection for resistance is reduced when low numbers of pathogen propagules present Fungicide coverage determines efficacy and minimizes pathogen survivors Heated solutions often are more effective Pathogen population is reduced. Resistance development to these salts is unlikely Accumulation of viable inoculum that may infect healthy fruit is prevented Exposure of pathogen to multiple fungicide classes reduces resistance frequency and potential Early detection of resistance increases the chance that its proliferation can be stopped

14 462 J.E. Adaskaveg and H. Förster Fig Plate technique for spore air sampling as fruits with high decay incidence are conveyed in bins from storage rooms for packing and shipping to markets. Fungicide-amended plates are exposed for approximately 1 2 min. Note white spray bar ( arrow ) for applying a chlorine sanitation solution to fruit as they are emptied from bins to the conveyer line to reduce airborne spores been developed that allow the rapid determination of EC 50 values of Penicillium species populations in packinghouses (Adaskaveg et al ; Kanetis et al ) or of individual isolates (Förster et al ). Spiral plating procedures are used in these methods where fungicide concentration gradients are created in agar plates using a spiral plater, and spore populations are either exposed to the plates in air samplings (Figs and 28.4a ) or individual isolates are streaked along the concentration gradients (Fig. 28.4b ). Sanitation of fruit, equipment, and fruit storage rooms with multisite, broadspectrum toxicants is one of the first strategies in resistance management that can be employed upon arrival of fruit at the packinghouse. Washing fruit with sanitizing oxidation treatments (e.g., sodium hypochlorite, peroxyacetic acid, ozonated water) and cleaning of equipment with oxidizers or quaternary ammonium compounds after or between handling fruit loads are ways broad-spectrum toxicants are utilized (Adaskaveg et al. 2002, 2004 ; Adaskaveg and Förster 2010 ). Proper sanitation reduces the amount of pathogen propagules exposed to the fungicide and thus reduces the chance for selection of resistance. The resistance frequency to any one fungicide in a pathogen population does not change, but reducing the total number of spores results in a proportional reduction in the level of resistant isolates present. Sanitation also includes the regulation of air flow and spore dispersal in the packinghouse by filtration to reduce the spore load and by directing the direction of air flow. Additionally, culled fruit should be removed from the packinghouse before sporulation occurs and should not be disposed of in a citrus orchard. Stored or packed fruits that were previously fungicide treated and that developed decay and pathogen sporulation should be sprayed with sodium hypochlorite (Fig ) and isolated in an area where the contaminated air can be exhausted away, and fruits should never be repacked at the same packinghouse location where newly

15 28 Resistance in Postharvest Pathogens of Citrus in the United States 463 Fig Monitoring methods for fungicide sensitivity in populations of Penicillium digitatum. Plates with radial fungicide concentration gradients (pyrimethanil was used in the plates shown) were exposed to spore masses during air samplings in a packinghouse ( a ) or were inoculated with spore suspensions of individual isolates along the concentration gradient ( b ). Plates were prepared using the spiral gradient dilution method and highest fungicide concentrations are toward the center and lowest concentrations toward the edge of the plate (Förster et al ). In ( a ), a high density of sensitive and resistant isolates is growing in a ring at lower concentrations, whereas numerous resistant colonies are growing at higher concentrations in the inner ring. In ( b ) three resistant isolates (replicated in opposite streaks) still grow at the highest concentrations, whereas five sensitive isolates are inhibited at lower concentrations harvested fruits are being processed. Any decay on treated fruit may be the result of selection of resistance to the fungicide that was used, and thus, the dissemination of surviving subpopulations should be minimized by physically separating the repacking process. Postharvest fungicides that inhibit sporulation of the pathogen on decaying escape fruit can further reduce pathogen population sizes in the packinghouse and minimize the chance of selection. Fungicides with anti-sporulation activity are imazalil and propiconazole (when decay is caused by strains sensitive to DMI compounds), as well as fludioxonil and azoxystrobin (Kanetis et al ). These treatments preferentially should be used for fruit being stored, whereas those without anti-sporulation activity should be used on packed fruit that are leaving the facility for marketing. Fungicides with single-site mode of action should not be used alone on a continuous basis. The use of rotations, mixtures, or pre-mixtures of compounds with different modes of action is an excellent strategy to reduce the risk of resistance development (Table 28.4 ). Ideally, rotations of mixtures or pre-mixtures should be used for fruit crops that are treated more than once, such as some citrus fruits (especially lemons) in California. Pre-mixtures are increasingly being registered for postharvest use in the United States, not only because of resistance management but also to increase the spectrum of activity of a treatment. Currently available pre-mixtures for citrus include imazalil/pyrimethanil (trade name, Philabuster), fludioxonil/thia-

16 464 J.E. Adaskaveg and H. Förster bendazole (Graduate Max), and azoxystrobin/fludioxonil (trade name, Graduate A+) (Table 28.1 ), but additional ones are in development such as fludioxonil/ azoxystrobin/propiconazole (Adaskaveg and Förster 2010 ). Unfortunately, in addition to the amounts of individual pesticides, recent regulatory restrictions in some export markets are limiting the total number of pesticides on a crop and thus impede the use of these anti-resistance strategies. Mixtures and pre-mixtures ideally should be used starting with the introduction of the new fungicide or before resistance is detected. This is because a pathogen population that is already resistant to one of the mixture components will be more easily selected for resistance to the second fungicide component. Mixtures should also be used in each application and at effective rates for each component in the mixture. Furthermore, each mixture component should have a similarly high efficacy and performance against target populations. References Adaskaveg JE (2004) Evaluation of new postharvest treatments to reduce postharvest decays and improve fruit quality in citrus packinghouse operations. Citrus Research Board, 2004 Annual Report, pp Adaskaveg JE, Förster H (2010) New developments in postharvest fungicide registrations for edible horticultural crops and use strategies in the United States. In: Prusky D, Gullino ML (eds) Post-harvest pathology: plant pathology in the 21st century: contributions to the 9th international congress. Springer, Dordrecht, pp Adaskaveg JE, Förster H, Sommer NF (2002) Principles of postharvest pathology and management of decays of edible horticultural crops. In: Kader A (ed) Postharvest technology of horticultural crops, 4th edn, Publication University of California Agriculture and Natural Resources, Oakland, pp Adaskaveg JE, Kanetis L, Soto-Estrada A, Förster H (2004) A new era of postharvest decay control in citrus with the simultaneous introduction of three new reduced-risk fungicides. Proc Int Soc Citric III: Barger WR (1928) Sodium bicarbonate as citrus fruit disinfectant. Calif Citrogr 13:164, Baudoin ABAM, Eckert JW (1982) Factors influencing the susceptibility of lemons to infection by Geotrichum candidum. Phytopathology 72: Brent KJ (1995) Fungicide resistance in crop pathogens: How can it be managed? FRAC monogr No. 1. GIFAP, Brussels Brent KJ, Hollomon DW (1998) Fungicide resistance: the assessment of risk, FRAC monogr. No. 2. GCPF, Brussels Brent KJ, Hollomon DW (2007) Fungicide resistance in crop pathogens: how can it be managed? 2nd edn, FRAC monogr No. 1. GCPF, Brussels Brown GE, Eckert JW (2000) Penicillium decays blue mold. In: Timmer LW, Garnsey SM, Graham JH (eds) Compendium of citrus diseases, 2nd edn. American Phytopathological Society Press, St. Paul, p 41 Cooke LR, Carlisle DJ, Donaghy C, Quinn M, Perez FM, Deahl KL (2006) The Northern Ireland Phytophthora infestans population characterized by genotypic and phenotypic markers. Plant Pathol 55: Davé BA, Kaplan HJ, Petrie JF (1980) The isolation of Penicillium digitatum Sacc. strains tolerant to 2-AB, SOPP, TBZ, and benomyl. Proc Fla State Hortic Soc 93:

17 28 Resistance in Postharvest Pathogens of Citrus in the United States 465 De Waard MA, Groeneweg H, Van Nistelrooy JGM (1982) Laboratory resistance to fungicides which inhibit ergosterol biosynthesis in Penicillium italicum. Neth J Plant Pathol 88: Eckert JW (1959) Lemon sour rot. Calif Citrogr 45:30 36 Eckert JW (1987) Biotypes of Penicillium digitatum with reduced sensitivity to imazalil. Phytopathology 77:1728 (Abstr) Eckert JW (1988) Dynamics of benzimidazole-resistant Penicillia in the development of postharvest decays of citrus and pome fruits. In: Delp CJ (ed) Fungicide resistance in North America. APS Press, St. Paul, pp Eckert JW, Eaks IL (1989) Postharvest disorders and diseases of citrus fruit. In: Reuther W, Calavan EC, Carman GE (eds) The citrus industry, volume V crop protection, postharvest technology, and early history of citrus research in California. Publication no University of California, Division of Agricultural and Natural Resources, Oakland, pp Förster H, Adaskaveg JE (2012) Fitness and competitiveness of Penicillium spp. isolates with reduced sensitivity to fludioxonil and pyrimethanil. Phytopathology 102(Suppl 6):S6.9 Förster H, Kanetis L, Adaskaveg JE (2004) Spiral gradient dilution, a rapid method for determining growth responses and EC 50 values in fungus-fungicide interactions. Phytopathology 94: Fritz R, Lanen C, Chapeland-Leclerc F, Leroux P (2003) Effect of the anilinopyrimidine fungicide pyrimethanil on the cystathionine β-lyase of Botrytis cinerea. Pestic Biochem Physiol 77:54 65 Ghosoph JM, Schmidt LS, Margosan DA, Smilanick JL (2007) Imazalil resistance linked to a unique insertion sequence in the PdCYP51 promoter region of Penicillium digitatum. Postharv Biol Technol 44:9 18 Hamamoto H, Hasegawa K, Nakaune R, Lee YJ, Akutsu K, Hibi T (2001) PCR-based detection of sterol demethylation inhibitor-resistant strains of Penicillium digitatum. Pest Manag Sci 57: Harding PR Jr (1962) Differential sensitivity to sodium orthophenylphenate by biphenyl-sensitive and biphenyl-resistant strains of Penicillium digitatum. Plant Dis Rep 46: Harding PR Jr (1972) Differential sensitivity to thiabendazole by strains of Penicillium italicum and P. digitatum. Plant Dis Rep 56: Harding PR Jr (1976) R23979, a new imidazole derivative effective against postharvest decay of citrus by molds resistant to thiabendazole, benomyl, and 2-aminobutane. Plant Dis Rep 60: Hewitt HG (1998) Fungicides in crop protection. CAB International, Cambridge, UK, 221 pp Holmes GJ, Eckert JW (1995) Relative fitness of imazalil-resistant and -sensitive biotypes of Penicillium digitatum. Plant Dis 79: Holmes GJ, Eckert JW (1999) Sensitivity of Penicillium digitatum and P. italicum to postharvest citrus fungicides in California. Phytopathology 89: Kader AA (2002) Postharvest technology of horticultural crops, 3rd edn, DANR Publication University of California, Oakland Kanetis L, Förster H, Adaskaveg JE (2007) Comparative efficacy of the new postharvest fungicides azoxystrobin, fludioxonil, and pyrimethanil for managing citrus green mold. Plant Dis 91: Kanetis L, Förster H, Adaskaveg JE (2008a) Baseline sensitivities for new postharvest fungicides against Penicillium spp. on citrus and multiple resistance evaluations in P. digitatum. Plant Dis 92: Kanetis L, Förster H, Jones CA, Borkovich KA, Adaskaveg JE (2008b) Characterization of genetic and biochemical mechanisms of fludioxonil and pyrimethanil resistance in field isolates of Penicillium digitatum. Phytopathology 98: Kanetis L, Förster H, Adaskaveg JE (2010) Determination of natural resistance frequencies in Penicillium digitatum using a new air-sampling method and characterization of fludioxoniland pyrimethanil-resistant isolates. Phytopathology 100: Kendall SJ, Hollomon DW (1998) Fungicide resistance. In: Hutson D, Miyamoto J (eds) Fungicidal activity: chemical and biological approaches to plant protection. Wiley, New York, pp

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