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Phytopathol. Mediterr. (2003) 42, 99112

Introduction

Alternaria species cause four distinct diseasesof citrus, namely, Alternaria brown spot of tange-rines and their hybrids, Alternaria leaf spot ofrough lemon, Alternaria black rot of fruit, and

Alternaria diseases of citrus Novel pathosystems

LAVERN W. TIMMER,1 TOBIN L. PEEVER,2 ZVI SOLEL3 and KAZUYA AKIMITSU4

1 Institute of Food and Agricultural Sciences, Citrus Research and Education Center,Department of Plant Pathology, University of Florida, Lake Alfred, FL 33850 USA

2 Department of Plant Pathology, Washington State University, Pullman, WA 99164-6430 USA3 Department of Plant Pathology, Agricultural Research Organization, The Volcani Center,

Bet Dagan 50250, Israel4 Laboratory of Plant Pathology, Department of Life Sciences, Faculty of Agriculture, Kagawa University,

Miki, Kagawa 761-0795 Japan

Summary. Citrus is affected by four diseases caused by Alternaria spp. Brown spot of tangerines, leaf spot of roughlemon, postharvest black rot of fruit occur widely in citrus areas of the world and are caused by different pathotypesof A. alternata. Mancha foliar occurs only on Mexican lime in western Mexico and is caused by A. limicola. Tangerineand rough lemon pathotypes produce host-specific toxins that affect membranes and respiration, respectively. Blackrot is always associated with wounds and is caused by most citrus-associated isolates of A. alternata that produceendopolygalacturonase. Alternaria brown spot is a serious disease of susceptible tangerines and their hybrids insemi-arid Mediterranean climates as well as in more humid areas. Conidia, produced on lesions on mature andsenescent leaves and stems under humid conditions, are dispersed by wind, and infect all juvenile tissues of suscep-tible cultivars when temperature and leaf wetness conditions are favorable. Commercially acceptable cultivars re-sistant to brown spot are being developed. Disease severity can be reduced by planting disease-free nursery stock onwider spacings, pruning tree skirts, and reducing irrigation and nitrogen fertilization. However, fungicides such asdithiocarbamates, triazoles, strobilurins, iprodione, or copper fungicides are used in most areas for disease control. Adisease-forecasting model, the Alter-Rater, has been developed in Florida to assist in timing fungicide sprays.

Key words: Alternaria alternata, Alter-Rater, disease models, toxins.

Mancha foliar on Mexican lime. The Alternariabrown spot pathogen tangerine pathotype affectsmany tangerines and their hybrids and produceslesions on immature fruit and leaves, induces leafand fruit drop, and produces spots and corky le-sions on mature fruit. The Alternaria leaf spotpathogen rough lemon pathotype produces sim-ilar symptoms on rough lemon leaves and pinpointlesions on fruit and affects only rough lemon andRangpur lime. The latter two pathogens producehost-specific toxins (HSTs) and the host range of

REVIEW

Corresponding author: L.W. TimmerFax: +1 863 956 4631E-mail: lwt@lal.ufl.edu

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each is very restricted (Kohmoto et al., 1979;1991). Alternaria black rot is a post-harvest dis-ease that occurs worldwide and produces inter-nal decay of all commercial citrus. The Alternariafungi causing the above diseases are small-spored,are morphologically similar, and all are consid-ered intra-specific variants of A. alternata (Peev-er et al., 2003). Pathogenicity tests, toxin assays,detection of toxin biosynthesis genes, or othergenetic markers are required to distinguish thesefungi. Mancha foliar is characterized by the pro-duction of small lesions on the leaves of Mexicanlime and a few other citrus varieties in westernMexico. This disease is caused by a large-sporedspecies, Alternaria limicola (Palm and Civerolo,1994) and is not associated with the productionof an HST.

The diseasesAlternaria brown spot of tangerines

The tangerine pathotype of A. alternata affectsmany tangerines and hybrids (Timmer et al.,2000a) and affects leaves, twigs and fruit. On youngleaves, the disease produces minute brown to blackspots. Symptoms can appear in as little as 24 hafter infection. Lesions usually continue to expandand large areas of the leaf may be killed by thehost-selective ACT-toxin (Kohmoto et al., 1993)even without tissue colonization. Chlorosis andnecrosis can extend along the veins as toxin istranslocated acropetally. On mature leaves, thedisease appears as distinct brown lesions surround-ed by a yellow halo (Fig. 1A). Affected leaves oftenabscise. Young shoots are also infected producingbrown lesions 1 to 10 mm in diameter. Infectedtwigs die back especially if the leaves have fallen.On fruit, brown to black lesions can vary fromminute spots to large crater-like lesions (Fig. 1D).Corky eruptions sometimes form and can be dis-lodged forming a pockmark on the surface. Severelyaffected fruit abscise reducing yield, and blemish-es on the remaining fruit greatly diminishing mar-ketability.

Alternaria leaf spot of rough lemon

This disease affects only rough lemon andRangpur lime, which are common rootstocks insome citrus-growing areas. Thus, this disease isonly commercially important in nurseries and

seed production blocks. Symptoms on leaves arevery similar to those produced on tangerines (Fig.1B) (Timmer et al., 2000a). The toxin producedby this pathotype is distinct from the tangerinepathotype (Kohmoto et al., 1979), and is calledACR-toxin or ACRL-toxin (Gardner et al., 1985;Nakatsuka et al., 1986a). Symptoms do not ap-pear on rough lemon leaves for about 3 days fol-lowing inoculation compared to 24 h for thebrown spot disease. Symptoms on fruit are mere-ly small brown specks (Timmer et al., 2000a) andare quite reduced relative to the tangerine patho-type.

Mancha foliar

Mancha foliar is a disease that primarily af-fects Mexican lime and occurs only in WesternMexico (Becerra et al., 1988; Timmer et al.,2000a). On Mexican lime, Mancha foliar produc-es small, reddish brown lesions on leaves thatare surrounded by chlorotic halos (Fig. 1C). Af-fected leaves often abscise and twigs may dieback. Small raised lesions are produced on fruit,but the symptoms disappear as the fruit devel-ops. Mancha foliar also occurs on grapefruit,navel oranges, and Tahiti lime, but seldom caus-es significant damage. Most other citrus is re-sistant to the disease.

Black rot

Black rot affects the central columella of thefruit and can affect all species of citrus (Brownand McCornack, 1972). External symptoms arenot often apparent and, if present, appear as asmall brown to black spot on the stylar end of thefruit (Brown and Eckert, 2000) (Fig. 1E). Affectedfruit are more brightly colored than normal fruitdue to ethylene generated in response to infec-tion. It appears that most small-spored isolatesof Alternaria are capable of causing black rot.These include saprophytic isolates colonizing deador senescent tissues, epiphytes from healthyleaves, as well as the tangerine and rough lemonpathotypes (Bhatia, Peever, and Timmer, unpub-lished). A wound or a natural crack is requiredfor penetration of the fungus. The ability to pro-duce endopolygalacturonase appears essential forisolates to cause black rot (Isshiki et al., 2001).None of the black rot strains tested to date pro-duce HST.

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The pathogens

Morphology, taxonomy, and classification

The first citrus-associated Alternaria speciesto be formally described was A. citri, the causalagent of citrus black rot (Pierce, 1902). Brownspot and rough lemon leaf spot pathogens weresubsequently identified as A. citri based on theirmorphological similarity to the black rot fungus

(Doidge, 1929; Ruehle, 1937; Kiely, 1964; Pegg,1966). However, the brown spot and leaf spot fun-gi are known to be biologically and pathological-ly distinct from the black rot fungi because theformer are able to infect young leaves and fruitand produce host-specific toxins (Kiely, 1964;Whiteside, 1976). The brown spot and leaf spotpathogens were considered A. alternata by Ko-hmoto et al. (1979) based on a published descrip-tion of conidial morphology and measurements

Fig. 1. A, Symptoms of Alternaria brown spot on mature Minneola tangelo leaves. B, Alternaria leaf spot symptomson rough lemon leaves. C, Symptoms of Mancha foliar on Mexican lime leaves (reprinted with permission from theCompendium of Citrus Diseases, 2nd ed., American Phytopathological Society, St. Paul, MN, USA). D, Symptoms ofAlternaria brown spot on Minneola tangelo fruit. E, Black rot symptoms on sweet orange fruit.

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of A. alternata (Simmons, 1967). Solel (1991) des-ignated the tangerine pathogen as A. alternatapv. citri. However this nomenclature does notaddress the status of the rough lemon pathogen.The terms tangerine pathotype and rough lem-on pathotype have been applied to denote theunique pathological attributes of the brown spotpathogen and rough lemon leaf spot pathogens,respectively, and we prefer these designations.Recent morphotaxonomic research has attempt-ed to clarify the identity of small-spored isolatesof Alternaria associated with brown spot and leafspot of citrus. One hundred and thirty-five iso-lates from the worldwide collection of L.W. Tim-mer and T.L. Peever, including isolates fromrough lemon leaf lesions and brown spot lesionson tangerines and tangerine hybrids, were ex-

amined and ten morphological species were de-scribed (Simmons, 1999), none of which was con-sidered representative of A. alternata or A. citri.

A phylogenetic analysis of small-spored, cit-rus-associated Alternaria isolates was recentlycompleted and included the ten morphologicalspecies recently described by Simmons (1999),several black rot isolates, and small-spored ref-erence species from other hosts (Peever et al.,2003). Using the ex-type isolates of Simmons(1999), it was possible to directly map the mor-phological species onto a phylogeny estimatedfrom a combined dataset consisting of a partialsequence of the coding region of an endopolyga-lacturonase (endoPG) gene (Isshiki et al., 2001;Peever et al., 2002) and two anonymous regionsof the genome. The analysis revealed eight well-supported clades which could be interpreted aseight phylogenetic species. The clades werebroadly congruent with the morphological spe-cies; however, three clades contained more thanone morphological species and one morphologi-cal species (A. citrimacularis) was polyphyletic.

Black rot isolates were distributed through-out the combined phylogeny in three clades. Oneblack rot isolate was found in the same phyloge-netic lineage as two saprophytic A. alternata iso-lates, another was found in a phylogenetic line-age with several brown spot and leaf spot iso-lates and a third was found in a lineage with A.arborescens, a host-specific toxin-producing path-ogen of tomato (Peever et al., 2003). These re-sults clearly demonstrate that phylogeneticallydistinct small-spored Alternaria taxa can be as-sociated with black rot and raise questions aboutthe validity of A. citri as a phylogenetic taxon.We find that many small-spored Alternaria spe-cies are able to cause black rot. Phylogeneticallydiverse isolates from black rot, brown spot andrough lemon leaf spot from citrus and additionalsmall-spored isolates from non-citrus hosts wereall able to induce black rot when inoculated intowounded citrus fruit (Bhatia, Peever and Tim-mer, unpublished). The lack of correlation be-tween phylogenetic lineage and unique pheno-typic, ecological or pathological characters amongthe small-spored citrus-associated Alternariaraises questions about the practical utility of boththe morphological species and species definedusing only phylogenetic criteria. The occurrence

Fig. 2. Scanning electron micrograph of conidiophoresof Alternaria alternata emerging from a stomata on amature leaf lesion.

Fig. 3. Conidia of Alternaria alternata (250).

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of multiple morphological species in severalclades and the polyphyly of at least one morpho-logical species clearly indicate that the morpho-logical species do not reflect evolutionary rela-tionships among these fungi. Until it can be dem-onstrated that unique ecological, biological orbiochemical characters can be associated with aspecific phylogenetic lineage, we advocate col-lapsing all small-spored, citrus-associated Alter-naria isolates, including brown spot, rough lem-on leaf spot and black rot isolates, into a singlespecies, A. alternata.

Mancha foliar is caused by A. limicola Simmonsand Palm, the only large-spored species of Alter-naria known to affect citrus (Palm and Civerolo,1994). In contrast to the non-pathogenic isolatesof Alternaria and those that cause brown spot, leafspot, or black rot, A. limicola is clearly distinguish-able morphologically and through molecular meth-ods (Peever et al., 2003). The conidia of this spe-cies are large (1622140190 m) with longbeaks (6090 m). A. limicola produces varioustoxins in culture, but they are not host specificas are the ACT and ACR-toxins (Becerra et al.,1988; Timmer et al., 2000a).

Host specificity

There is a high degree of host specificity amongisolates from tangerine and those from rough lem-on (Kohmoto et al., 1979, 1991; Peever et al., 1999).In Florida, the vast majority of the isolates col-lected from Minneola tangelo were pathogenic tothat host and only 3% were non-pathogenic andnone was pathogenic to rough lemon (Peever etal., 1999). Most isolates from rough lemon werepathogenic on the host of origin, but a few werepathogenic on Minneola tangelo and not roughlemon. A substantial portion, 44%, were not path-ogenic to either host.

When disease symptoms were first found ongrapefruit and on Sunburst tangerine in Florida,the possibility of host specificity within the tan-gerine pathotype was raised (Timmer and Peev-er, 1997). Using random amplified polymorphicDNA, isolates from grapefruit and the tangerinehybrid Nova could be distinguished from thosefrom Robinson, Sunburst, Minneola, Orlando, andMurcotts (Peever et al., 2000). However, cross in-oculation studies on the different hosts did notsupport the host specificity seen with molecular

markers. In the inoculations, Minneola was con-sistently the most susceptible followed by Orlan-do, Sunburst, Nova, and grapefruit in decreasingorder of susceptibility regardless of the source hostof the isolates.

Disease cycle and epidemiology

Alternaria brown spot of tangerine is the onlyAlternaria disease of citrus for which there is anappreciable amount of information regarding ep-idemiology. Since leaf spot of rough lemon is rel-atively unimportant commercially, the ecologyand epidemiology of this pathogen have been lit-tle studied. The disease cycle is simple sincethere is no teleomorph known for A. alternata(Timmer, 1999). Conidia are produced primarilyon the surface of lesions on mature or senescentleaves (Fig. 2,3) and on blighted twigs. Relative-ly few, if any, are produced on young lesions onleaves or mature lesions on fruit. Conidium pro-duction is greatest when leaves are lightly mois-tened or held at high humidity, with fewer pro-duced where leaves are very wet (Timmer et al.,1998). Almost no conidia are formed at low ormoderate relative humidities if leaves are freeof moisture. Conidium production, dispersal, andinfection are presumed to be similar to those ofthe tangerine pathotype. Likewise control meas-ures have not been established for this disease.

Release of conidia from sporulating brownspot lesions is triggered by rainfall or by suddenchanges in relative humidity (Timmer et al.,1998). Rainfall is probably most important forspore release in humid areas such as Florida (US)or Brazil. However, in Mediterranean areaswhere little rain falls during the susceptible pe-riod, spore release may be triggered by a sharpdrop in relative humidity (RH) when the dewdries. Spores are dispersed by wind currents andare eventually deposited on the surface of sus-ceptible tissues. With dew the following night,the conidia germinate and eventually infect theleaves or fruit. Penetration of the leaf can occurdirectly or through stomata and in studies in Is-rael is consistently associated with appressoriaformation (Solel and Kimchi, 1998). Preliminaryobservations in Florida indicate that penetrationoccurs through stomata on the undersurface ofthe leaf without appressorium formation (Bha-

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tia et al., 2002).The optimum temperature for infection is

27C (Canihos et al., 1999). As temperatures de-cline, longer wetting periods are needed for in-fection to occur. At 32C, little infection occurseven with ample moisture. Small amounts of in-fection can occur with leaf wetness durations of48 h, but usually 1012 h of wetness are need-ed for substantial infection (Canihos et al., 1999;Timmer et al., 2000b). In many semi-arid areas,temperatures are cool at night when dew occursand may be below the optimum. Thus longerwetting periods may be required for infection. Aswith Alternaria spp. from other crops, germinat-ed conidia may survive the heat of the day andcontinue development the following night (Ro-tem, 1994). In humid areas such as Florida, Co-lombia, or Brazil, temperatures following rain-fall may be near the optimum during the periodof susceptibility.

In Mexico, Mancha foliar occurs primarily inthe dry, cool winters and diminishes with warm,wet weather in summer (Stapleton and Garcia-Lopez, 1988). Conidia germinate in 4 h and in-fect leaves in about 12 h. In the field, infectionlevels are highest when temperatures drop be-low 24C. Sporulation begins about one week af-ter the symptoms appear.

Dithiocarbamate fungicides are effective fordisease control. Applications are needed one weekafter shoot emergence and again two weeks lat-er (Timmer et al., 2000a).

In the field, black rot occurrence is sporadic.The disease is more common in semi-arid areasbecause Alternaria is more prevalent as an epi-phyte, endophyte, and saprophyte there than inhumid areas. It occurs commonly in navel orang-es because of natural openings created by growthof the navel (Brown and Eckert, 2000). It is alsofrequently observed in lemons, especially wherefruit is held in cold storage for long periods. Pre-and post-harvest fungicide applications providevery little control of the disease. If black rot oc-curs in the grove, harvest must be delayed untilmost of the affected fruit has fallen. Hormones,such as 2, 4-D applied postharvest, can delay se-nescence and reduce the incidence of the disease.

Geographical distribution and diversity

Alternaria brown spot disease was first re-

ported on Emperor mandarin in Australia in 1903(Cobb, 1903) and the causal agent was identi-fied as a species of Alternaria in 1959 after anumber of organisms were investigated as pos-sible pathogens (Kiely, 1964; Pegg, 1966). Thedisease later appeared in the USA (Whiteside,1976), and now occurs in Israel (Solel, 1991),South Africa (Schutte et al., 1992), Turkey (Cani-hos et al., 1997), Spain (Vicent, 2000), and Bra-zil and Argentina (Goes et al., 2001; Peres et al.,2003). Due to the morphological similarity be-tween the brown spot and black rot pathogens,the former was originally identified as A. citriEllis and Pierce (Pegg, 1966; Whiteside, 1976), afungus that had been first described as the causeof citrus black rot. Alternaria leaf spot of roughlemon was first reported from South Africa(Doidge, 1929). Alternaria black rot, also knownas stem-end rot, was reported as early as the1900s in California (Pierce, 1902; Roger and Ear-le, 1917; Bartholomew, 1926; Bliss and Fawcett,1944).

Isolates of A. alternata were collected fromMinneola tangelo in the United States, Colom-bia, Australia, Israel, Turkey and South Africato infer the worldwide population structure andphylogeography of the pathogen (Peever et al.,2002). Both RAPD markers and sequence datafrom an endoPG revealed that isolates from Flor-ida and Colombia were distinct from isolatessampled in the other parts of the world. The en-doPG data separated isolates into three phylo-genetic lineages which correlated to country oforigin. Isolates from the United States, Austral-ia, South Africa and Israel were found in a sec-ond clade whereas isolates from Australia, Isra-el, South Africa, and Turkey were found in a thirdclade. Peever et al. (2003) speculated that thesethree phylogenetic lineages were introduced in-dependently into each citrus-growing region froma common source population on plant material.This source population may have been SoutheastAsia, the center of origin of citrus, although lit-tle is known about the disease or the pathogenin this region. Isolates were tested for patho-genicity on detached leaves of Minneola tangeloand the Florida strains were significantly morevirulent than strains from other locations. Iso-lates from Florida may be more virulent becausethey have more copies of genes controlling toxin

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biosynthesis (ACTT) and produce more ACT-toxinthan isolates from other locations. A small per-centage of the worldwide isolates were identifiedas being non-pathogenic or having greatly re-duced virulence and these isolates are currentlybeing screened for toxin production and the pres-ence of ACTT sequences.

PathogenesisBrown spot and leaf spot diseases

The host specificity of the tangerine and roughlemon pathotypes of A. alternata depends uponthe production of HSTs that possess the same se-lectivities as the pathogens (Kohmoto et al., 1979,1991). The toxin from the rough lemon patho-type was named ACR or ACRL-toxin, and thatfrom the tangerine pathotype was named ACT-toxin. The structure of the major form of ACR-toxin, ACRL-toxin I (MW 496), was character-ized as a dihydropyrone ring with a polyalcoholside chain (Gardner et al., 1985; Nakatsuka etal., 1986a). ACR-toxin causes water congestion,veinal necrosis (Kohmoto et al., 1979; Akimitsuet al., 1994), inhibition of 14C-proline incorpora-tion (Gardner et al., 1985), and induces a rapidincrease of electrolyte leakage (Kohmoto et al.,1979; Akimitsu et al., 1994) only on the toxin-sensitive citrus species, rough lemon and Rang-pur lime. Interestingly, light irradiation can sup-press toxin-induced necrosis as well as electro-lyte leakage (Akimitsu et al., 1994), and a darkperiod of more than 3 h during the light irradia-tion overcomes the suppressive effect of light(Akimitsu et al., 1994).

The major form of HST produced by the tan-gerine pathotype was designated as ACT-toxin I(Kohmoto et al., 1993). The structure of ACT-tox-in is closely related to AK- and AF-toxins, whichare the HSTs produced by the Japanese pear andstrawberry pathotypes of A. alternata, respec-tively (Nakashima et al., 1985; Nakatsuka et al.,1986b, 1990; Kohmoto et al., 1993). These toxinsshare a common 9,10-epoxy-8-hydroxy-9-methyl-decatrienoic acid moiety (Nakashima et al., 1985;Nakatsuka et al., 1986b; Kohmoto et al., 1993).ACT-toxin causes veinal necrosis and a rapid in-crease in electrolyte loss from susceptible leaves,but the toxin has no effect on resistant leaves(Kohmoto et al., 1993). The mode of action of ACT-

toxin is still uncertain, but a rapid loss of elec-trolytes from leaf tissues and ultrastructuralchanges of cells treated with the toxin indicatedthat the primary action site of ACT-toxin waslikely the plasma-membrane (Kohmoto et al.,1993).

A cluster of genes controlling biosynthesis ofACT-toxin was identified using heterologousprobes of AKT sequences which control biosyn-thesis of a 9,10-epoxy-8-hydroxy-9-methyl-deca-trienoic acid moiety in AK-toxin from the Japa-nese pear pathotype of A. alternata (Tanaka etal., 1999). Portions of these genes used as heter-ologous probes detected homologous in severalisolates of A. alternata tangerine pathotype butnot from isolates that do not produce ACT-toxinsuch as the rough lemon pathotype, saprophytes,or black rot isolates (Masunaka et al., 2000).

The target site of ACR-toxin was identified asthe mitochondrion. Electron microscopic exami-nation of the toxin-treated cells showed that 10to 18% of the mitochondria were disrupted with-in one hour after treatment with the toxin. Cristawere swollen, fewer in number and vesiculatedwith a lower matrix density. Disruption of mito-chondrial functions by ACR-toxin was also ex-amined by monitoring an oxidative-phosphoryla-tion and mitochondrial membrane potential us-ing isolated physiologically active citrus mito-chondria (Akimitsu et al., 1989). ACR-toxincaused uncoupling similar to classic protono-phores, such as 2,4-dinitrophenol or carbonylcyanide m-chlorophenyl hydrazone, with a lossof membrane potential, but the effects differedslightly from other uncouplers because the toxinalso causes leakage of co-factor, NAD+ from thetricarboxylic acid cycle (Akimitsu et al., 1989).These effects of ACR-toxin are specific to mito-chondria isolated from susceptible cultivars.Dancy tangerine, Emperor mandarin, and grape-fruit, which are susceptible to ACT-toxin, arecompletely insensitive to ACR-toxin I (Akimitsuet al., 1989).

Defense responses of citrus inoculated withAlternaria leaf spot pathogens have not been ex-amined in detail to date. The expression patternsof several defense-related genes including lipox-ygenase (RlemLOX) (Gomi et al., 2002a), hyper-peroxide lyase (Gomi et al., 2003), chalcone syn-thase (Gotoh et al., 2002; Nalumpang et al.,

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2002a), polygalacturonase-inhibiting protein(Gotoh et al., 2002; Nalumpang et al., 2002a,2002b), chitinases (Gomi et al., 2002b) genes havebeen characterized in rough lemon in responseto these pathogens. All these genes are highlyinducible in rough lemon leaves by infection withnonpathogenic A. alternata, and expression ofthese genes was detected within 2 h after wound-ing or inoculation of rough lemon leaves withconidia of the A. alternata tangerine pathotype(isolate SH20), which is pathogenic to tangerinesand mandarins but not to rough lemon (Gomi etal., 2002a, 2002b; Gotoh et al., 2002; Nalumpanget al., 2002a, 2002b). Another nonpathogenicstrain of A. alternata (isolate O-94), which is notpathogenic to any citrus tested (Akimitsu et al.,1989; Kohmoto et al., 1991), also induced expres-sion of these genes within 2 h, but the intensityof the bands was not as strong as those inducedby SH20. Because there are no apparent mor-phological or biochemical differences betweenSH20 and O-94 except that SH20 produces ACT-toxin (Kohmoto et al., 1979, 1991, 1993; Masu-naka et al., 2000), the toxin might have a role aselicitor in the greater accumulation of the tran-scripts. In contrast, induction of these defense-related gene expression was delayed or sup-pressed when pathogenic A. alternata rough lem-on pathotype (AC325) was inoculated on roughlemon leaves (Gomi et al., 2002a, 2002b; Gotohet al., 2002; Nalumpang et al., 2002a, 2002b).This system, using either an HST-producing ora non-producing A. alternata strain, which ledto a clear susceptible or resistant response inrough lemon leaves, may be a good model for fur-ther evaluation of the role of other defense-re-lated genes including PR-proteins.

Alternaria black rot disease

Although the black rot pathogen has beenknown to produce several toxins such as tenua-zonic acid, alternariol methyl ether, and alter-nariol (Logrieco et al., 1990), these toxins haveno known role in pathogenesis. The pathogenic-ity of this fungus depends upon production of anextracellular enzyme that can degrade pectic pol-ymers in cell walls during infection. The possi-ble role of cell wall-degrading enzymes in patho-genicity, including penetration, maceration, nu-trient acquisition, plant defense induction, andsymptom expression have been investigated

(Cooper, 1983, 1984; Walton, 1994). The endoPGshave been purified and the genes cloned fromboth a black rot pathogen and a rough lemonpathotype isolate (Isshiki et al., 1997, 2001). Thesequences of these genes and biochemical char-acteristics of the enzymes they encode are high-ly similar. However, these genes in the respec-tive pathogens were disrupted by gene target-ing, the phenotypes of the mutants were com-pletely different (Isshiki et al., 1997, 2001). AnendoPG mutant of the black rot pathogen wassignificantly reduced in its ability to cause blackrot symptoms and penetration into citrus fruitsas well as in the maceration of potato tissue andcould not colonize citrus peel segments (Isshikiet al., 2001). In contrast, an endoPG mutant ofthe rough lemon pathotype was unchanged inpathogenicity on rough lemon leaves (Isshikiet al., 2001). Thus, an endoPG was not requiredfor pathogenicity of the rough lemon pathotypeand it played different roles in the pathogenici-ty of these two closely related fungi.

Disease controlHost resistance

Relatively few citrus species and cultivars aresusceptible to Alternaria brown spot and most ofthe susceptibility occurs in the progeny of Dan-cy tangerine. Many of the susceptible cultivarssuch as Minneola, Orlando, Nova, Lee, and Sun-burst have Dancy tangerine in their parentage.The disease occurs on other varieties not knownto have Dancy in their background such as Mur-cott, Emperor, and Ponkan. Alternaria brownspot has been observed on Ellendale and Idithmandarins, calamondin, and red grapefruit inIsrael (Solel and Kimchi, 1997) and on grapefruitin Florida (Timmer and Peever, 1997). Brownspot in grapefruit is associated usually with se-vere disease in nearby plantings of susceptibletangerines and their hybrids. Many other citrusspecies may be infected by artificial inoculationor are affected by the toxin but are not affectedin the field (Kohmoto et al., 1979; Solel and Kim-chi, 1997).

Based on the pattern of susceptibility of vari-ous citrus cultivars and hybrids, Kohmoto et al.(1991) concluded that the susceptibility was in-herited from the Dancy parent as a dominant

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trait. Dalkilic (1999) found that segregation froma cross of Clementine tangerine, a resistant spe-cies, and a Clementine Minneola hybrid pro-duced a 1:1 ratio of susceptible:resistant proge-ny. The reciprocal cross, however, yielded a 3:1ratio of resistant to susceptible offspring. He con-cluded that resistance was recessive but the phe-notype may have been affected by cytoplasmicallyinherited paternal traits. Dalkilic (1999) identi-fied RAPD markers that were consistently asso-ciated with resistance.

Dancy tangerine is the source of much of thesusceptibility to Alternaria brown spot and wasselected in Florida and used as a parent in muchof the breeding program for new tangelos andmore complex hybrids. Interestingly, Alternariabrown spot appeared in Australia on Emperormandarin (Cobb, 1903) long before it was de-scribed in Florida (Whiteside, 1976). Brown spothas also appeared on Ponkan mandarin in Bra-zil (Goes et al., 2001) but little is known aboutthe susceptibility of this cultivar. Molecular phy-logenetic studies of the brown spot pathogens col-lected from around the world indicate that theFlorida population is ancestral to the other pop-ulations (Peever et al., 2002). The authors spec-ulate that the pathogen originated with citrusin southeast Asia and the most ancestral popu-lation was introduced into Florida. However, thepossibility remains that the citrus pathogen mayhave originated elsewhere from other toxin-pro-ducing Alternaria populations on other hosts.

Since relatively few cultivars are susceptibleto brown spot, selection or production of commer-cially acceptable, brown-spot resistant cultivarsseems feasible. This could be achieved by classi-cal breeding or induced in various existing culti-vars by molecular techniques, irradiation, or in-duced mutagenesis. An Alternaria-tolerant cul-tivar similar to Minneola will be released soonfrom the University of Florida (F.G. Gmitter,personal communication).

Cultural practices

There is little research on the effects of cul-tural practices on the severity of brown spot in-fection. However, studies of the effect of environ-mental factors that affect disease severity (Cani-hos et al., 1997; Timmer et al., 1998, 2000b) pro-vide some information to suggest certain cultur-

al practices which might be helpful. Avoidanceof overhead irrigation and use of under-tree irri-gation systems in Florida has reduced diseaseseverity in some groves. Wider spacing and skirt-ing of trees allow better ventilation and seem toreduce disease severity. Avoidance of excess irri-gation and nitrogen fertilization have been rec-ommended for some time to avoid production oflarge amounts of susceptible tissue (Whiteside,1976; Timmer et al., 2001). While perhaps help-ful, it is difficult to reduce irrigation and fertili-zation enough without jeopardizing fruit produc-tion. Scheduling of hedging and topping just pri-or to the dry season in Florida allows develop-ment of the consequent flush of growth with aminimum of infection and inoculum production.

Planting of new groves with Alternaria-freenursery stock has been helpful in reducing brownspot in the early years (Timmer, 2003). Grovesinitiated using healthy stock in Florida have re-mained relatively disease-free for a surprisinglylong time, sometimes up to 68 years, even wheninoculum is present in vicinity. This may indi-cate that long distance dispersal of conidia oc-curs rarely. Healthy nursery stock can be pro-duced by selecting budwood from healthy moth-er plants and then growing trees in greenhouseswith sub-irrigation (Timmer, 2003). While cul-tural measures alone are seldom sufficient forcommercial control, they can greatly reduce in-oculum levels and disease severity and enhancethe efficiency of fungicide control programs.

Chemical control

Foliar fungicide applications are usually nec-essary to produce fruit with good external qual-ity in areas where Alternaria brown spot is com-mon. Depending on the climate in different are-as, from 3 to 15 applications may be needed. Inthe early years after discovery of the disease inFlorida, captafol was widely used and highly ef-fective for disease control (Whiteside, 1976; Tim-mer and Zitko, 1997). Since captafol has a longresidual and is redistributed, few applicationswere needed for good disease control. However,this product is no longer registered in most are-as due to health concerns. Iprodione is also veryeffective for disease control (Timmer and Zitko,1992, 1994, 1997; Solel et al., 1997) but resist-ance developed in Israel (Solel et al., 1996) limits

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L.W. Timmer et al.

its usefulness in some groves. Copper fungicidesare widely used for Alternaria control in Florida(Timmer et al., 2003) and when applied on a time-ly basis provide good control of the disease. How-ever, copper products cause stippling of the fruitwhen applied at high temperatures and must beused with caution. Other fungicides that are ef-fective and registered in Israel are the dithio-carbamates, triazoles, and famoxadon (Sadowskyet al., 2002a). Recently the strobilurin fungicideshave been evaluated and proven effective for con-trol of brown spot (Bhatia et al., 2002a, 2002b;Sadowsky et al., 2002a; Timmer, 2002). Azoxys-trobin and pyraclostrobin are generally more ef-fective than trifloxystrobin. Strobilurins are sin-gle site of action fungicides (Sierotski et al.,2000a, 2002b) and thus, prone to developmentof resistance and must be alternated or mixedwith other products.

Numerous products that induce resistance inthe host have been evaluated for control of Al-ternaria brown spot (Agostini et al., 2003). Prod-ucts containing or producing phosphites or sali-cylic acid significantly reduce disease severity.These materials have some promise in a program

alone or perhaps in combination with standardfungicides (Sadowsky et al., 2002b).

Fruit must be protected from petal fall untilabout mid-summer, but some infection may oc-cur after that time. Disease levels are generallylow and lesions usually small, but some largeblack lesions can occur as a result of late infec-tion in Israel and may induce fruit drop (Sad-owsky et al., 2002a). In addition it is often nec-essary to protect spring flush foliage to preventbuild-up of high levels of inoculum prior to fruitset. In high rainfall areas, it may not be possibleto control disease adequately. In Colombia, asmall, but highly profitable production of Min-neola tangelos was eliminated when Alternariawas introduced on nursery stock from Florida.Production of high quality fruit is also difficultin Florida and Brazil now that A. alternata iswell-established in those areas.

The Alter-Rater, a system for timing of fungi-cide application was developed in Florida (Tim-mer et al., 2000b). The factors used in the modelare: 1) the occurrence of rainfall over 2.5 mm;disease severity is related to the occurrence butnot the amount of rainfall; 2) the total hours of

Table 1. The daily scores assigned by the Alter-Rater model to various combinations of rainfall, leaf wetness, andtemperature (reprinted with permission from Bhatia et al., 2003).

Rainfall >25 mm Leaf wetness >10 h Average daily Assigned scoreatemperature (C)

+ + 2028 11+ + >28 8+ + 28 4+ - 28 6- + 28 0- -

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Alternaria diseases of citrus Novel pathosystems

leaf wetness; about 10 h are needed for signifi-cant infection, and 3) average daily temperature;temperature between 20 and 32C are optimal.Points are assigned to each day based on theweather and are accumulated until a preassignedthreshold is reached and then an application offungicide is made (Table 1). Thresholds are as-signed based on the disease history in the groveand the susceptibility of the cultivar. The sys-tem has proven successful in Florida as long asthe threshold is properly selected (Bhatia et al.,2003). The system also appears to be function-ing well in Brazil (N.A.R. Peres and L.W. Tim-mer, unpublished). In semi-arid areas where norainfall occurs after bloom, the system is likelyto be less useful. Since temperatures and dewperiods are often uniform from day to day, tim-ing of spray would probably not differ substan-tially from a calendar spray program.

Concluding remarks

Alternaria diseases of citrus represent someinteresting pathosystems. The tangerine patho-type produces a host-specific toxin and is closelyrelated to similar fungi that affect Japanese pear,apple, strawberry, and other hosts. The roughlemon pathotype produces another host-specifictoxin which differs chemically as well as in itsmode of action from the tangerine pathotype. Thegenes for toxin production are located on a veryshort chromosome in the pathogen. The evolu-tionary history of these pathogens could be fas-cinating if it can be elucidated. Alternaria alter-nata is a common saprophyte on citrus leaves inthe grove, but these isolates are able to causeblack rot of fruit after harvest. Such saprophytesare commonly isolated from leaf lesions of roughlemon, but rarely from Minneola tangelo. Theecological relationship of the various isolates onthe leaf surface needs further investigation toelucidate the roles of different isolates of thesame species. In contrast to many citrus diseas-es, the development of resistant cultivars by clas-sical breeding or genetic manipulation appearsfeasible. Resistance of citrus to Alternaria brownspot is inherited as a recessive trait. Furtherinvestigation of the epidemiology, fungicide ac-tivity, and timing should improve disease con-trol on the short-term.

Acknowledgments

This research was supported by the FloridaAgricultural Experiment Station, and approved forpublication as Journal Series No. N-02402.

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Accepted for publication: July 21, 2003