Pathotypes and genetic relationship of worldwide collections of Elsinoë spp. causing scab diseases of citrus.

Two scab diseases are recognized currently on citrus: citrus scab, caused by Elsinoë fawcettii, and sweet orange scab, caused by E. australis. Because the two species cannot be reliably distinguished by morphological or cultural characteristics, host range and molecular methods must be used to identify isolates. Four pathotypes of E. fawcettii and two of E. australis have been described to date based on host range. The host specificity and genetic relationships among 76 isolates from Argentina, Australia, Brazil, Korea, New Zealand, and the United States were investigated. Based on pathogenicity tests on eight differential hosts, 61 isolates were identified as E. fawcettii and 15 as E. australis. Of 61 isolates of E. fawcettii, 24 isolates were identified as the Florida broad host range (FBHR) pathotype, 7 as the Florida narrow host range (FNHR) pathotype, 10 as the Tryon's pathotype, and 3 as the "Lemon" pathotype. Two new pathotypes, the "Jingeul" and the satsuma, rough lemon, grape-fruit, clementine (SRGC), are described, and four isolates did not fit into any of the known pathotypes of E. fawcettii. Of the 15 isolates of E. australis from Argentina and Brazil, 9 belonged to the sweet orange pathotype and 6 from Korea to the natsudaidai pathotype. E. fawcettii and E. australis were clearly distinguishable among groups by random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) assays and the E. fawcettii group was divided into three subgroups, A-1, A-2, and A-3. The A-1 group was composed of the FBHR, FNHR, and SRGC pathotypes; some Lemon pathotypes; and the uncertain isolates. The A-2 subgroup included all of the Tryon's pathotype isolates and one of the three Lemon pathotype isolates and the A-3 group contained the Jingeul pathotype isolates. E. australis was differentiated into two groups: B-1, the natsudaidai pathotype isolates, and B-2, the sweet orange pathotype isolates. Isolates of E. fawcettii and E. australis were clearly distinguishable by sequence analysis of the internal transcribed spacer (ITS) region and the translation elongation factor 1 alpha (TEF) gene. There were also fixed nucleotide differences in the ITS and TEF genes that distinguished subgroups separated by RAPD-PCR within species. We confirmed two species of Elsinoë, two pathotypes of E. australis, and at least six pathotypes of E. fawcettii and described their distribution in the countries included in this study.

Host range and genetic relatedness of Colletotrichum acutatum isolates from fruit crops and leatherleaf fern in Florida.

Isolates of Colletotrichum acutatum were collected from anthracnose-affected strawberry, leatherleaf fern, and Key lime; ripe-rot-affected blueberry; and postbloom fruit drop (PFD)-affected sweet orange in Florida. Additional isolates from ripe-rot-affected blueberry were collected from Georgia and North Carolina and from anthracnose-affected leatherleaf fern in Costa Rica. Pathogenicity tests on blueberry and strawberry fruit; foliage of Key lime, leatherleaf fern, and strawberry; and citrus flowers showed that isolates were highly pathogenic to their host of origin. Isolates were not pathogenic on foliage of heterologous hosts; however, several nonhomologous isolates were mildly or moderately pathogenic to citrus flowers and blueberry isolates were pathogenic to strawberry fruit. Based on sequence data from the internal transcribed spacer (ITS)1-5.8S rRNA-ITS2 region of the rDNA repeat, the glutaraldehyde-3-phosphate dehydrogenase intron 2 (G3PD), and the glutamine synthase intron 2 (GS), isolates from the same host were identical or very similar to each other and distinct from those isolated from other hosts. Isolates from leatherleaf fern in Florida were the only exception. Among these isolates, there were two distinct G3PD and GS sequences that occurred in three of four possible combinations. Only one of these combinations occurred in Costa Rica. Although maximum parsimony trees constructed from genomic regions individually displayed little or no homoplasy, there was a lack of concordance among genealogies that was consistent with a history of recombination. This lack of concordance was particularly evident within a clade containing PFD, Key lime, and leatherleaf fern isolates. Overall, the data indicated that it is unlikely that a pathogenic strain from one of the hosts examined would move to another of these hosts and produce an epidemic.

Postbloom fruit drop of citrus and key lime anthracnose are caused by distinct phylogenetic lineages of Colletotrichum acutatum.

Colletotrichum acutatum causes two diseases of citrus, postbloom fruit drop (PFD) and Key lime anthracnose (KLA). PFD is a disease restricted to flowers of sweet orange and most other citrus, and symptoms include petal necrosis, abscission of developing fruit, and the formation of persistent calyces. KLA is a disease of foliage, flowers, and fruits of Key lime only, and symptoms include necrotic lesions on leaves, fruits, twigs, flowers, and blight of entire shoots. The internal transcribed spacers 1 and 2 and the gene encoding the 5.8S ribosomal RNA subunit within the nuclear ribosomal cluster (ITS) and intron 2 of the glyceraldehyde-3-phosphate dehydrogenase gene (G3PD) were sequenced for isolates from PFD-affected sweet orange and KLA-affected Key limes collected in the United States (Florida), Brazil (São Paulo), Mexico, Belize, Costa Rica, and the Dominican Republic to determine if there are consistent genetic differences between PFD and KLA isolates over the geographic area where these diseases occur. Based on the sequence data, isolates clustered into two well-supported clades with little or no sequence variation among isolates within clades. One clade (PFD clade) contained PFD isolates from all countries sampled plus a few isolates from flowers of Key lime in Brazil. The other clade (KLA clade) contained KLA isolates from Key lime foliage from all countries sampled and one isolate from flowers of sweet orange in Mexico. In greenhouse inoculations with PFD and KLA isolates from Florida, isolates from both clades produced PFD symptoms on Orlando tangelo flowers, but KLA-clade isolates produced significantly less severe symptoms. PFD-clade isolates were not pathogenic to Key lime foliage, confirming previous studies. The differentiation of PFD and KLA isolates into two well-supported clades and the pathogenicity data indicate that PFD and KLA are caused by distinct phylogenetic lineages of C. acutatum that are also biologically distinct. PFD is a recently described disease (first reported in 1979) relative to KLA (first reported in 1912) and it had been proposed that strains causing PFD evolved from strains causing KLA eventually losing pathogenicity to Key lime foliage. We reject the hypothesis that PFD strains have diverged from KLA strains recently based on estimated divergence times of haplotypes and it appears that PFD and KLA strains have been dispersed throughout the Americas independently in association with each host.

Selection for Pathogenicity to Strawberry in Populations of Colletotrichum gloeosporioides from Native Plants.

ABSTRACT Colletotrichum gloeosporioides causes a serious crown rot of strawberry and some isolates from native plants are pathogenic to strawberry. C. gloeosporioides from lesions on wild grape and oak were sampled at two sites adjacent to commercial strawberry fields in Florida and two distant sites. Random amplified polymorphic DNA (RAPD) marker data and restriction enzyme digests of amplified rDNA were used to determine whether isolates were from the same C. gloeosporioides subgroup that infects strawberry. There were 17 to 24 native host isolates from each site that clustered with a group of strawberry crown isolates based on RAPD markers. Among strawberry isolates, there were two rDNA genotypes identified by restriction enzyme analysis. Both genotypes were present among native host isolates sampled from all four sites. There was some evidence that the different rDNA genotypes differentiated two closely related subpopulations, although the proportion of pathogenic isolates from native hosts among the two different genotypes was not different. The incidence of isolates pathogenic to strawberry was greater at sites close to strawberry fields relative to sites distant from strawberry fields for isolates with a BstUI(-)/MspI(+) rDNA genotype (44 versus 13%), a BstUI(+)/MspI(-) genotype (57 versus 16%), or when both genotypes were analyzed together (46 versus 15%). Based on these results, it appears that the C. gloeosporioides subgroup that causes crown rot on strawberry is widely distributed in Florida and that selection for pathogenicity on strawberry occurs in the area where this host is grown in abundance.

Development of PCR Assays for the Identification of Species and Pathotypes of Elsinoë Causing Scab on Citrus.

Two scab pathogens of citrus, Elsinoë fawcettii and E. australis, cause citrus scab and sweet orange scab, respectively, and pathotypes of each species have been described. The two species cannot be readily distinguished by morphological or cultural characteristics and can be distinguished only by host range and the sequence of the internal transcribed spacer (ITS) region. In this study, random amplified polymorphic DNA (RAPD) assays clearly distinguished E. fawcettii and E. australis, and the sweet orange and natsudaidai pathotypes within E. australis also could be differentiated. We developed specific primer sets, Efaw-1 for E. fawcettii; Eaut-1, Eaut-2, Eaut-3, and Eaut-4 for E. australis; and EaNat-1 and EaNat-2 for the natsudaidai pathotype within E. australis using RAPD products unique to each species or pathotype. Other primer sets, Efaw-2 and Eaut-5, which were specific for E. fawcettii and E. australis, respectively, were designed from previously determined ITS sequences. The Efaw-1 and Efaw-2 primer sets successfully identified E. fawcettii isolates from Korea, Australia, and the United States (Florida) and the Eaut-1 to Eaut-5 primer sets identified both the sweet orange pathotype isolates of E. australis from Argentina and the natsudaidai pathotype isolates from Korea. The EaNat-1 and EaNat-2 primer sets were specific for isolates of the natsudaidai pathotype. The Efaw-1 and Efaw-2 primer sets successfully detected E. fawcettii from lesions on diseased leaves and fruit from Korea and primer pairs Eaut-1, Eaut-2, Eaut-3, Eaut-4, and Eaut-5 detected E. australis from lesions on sweet orange fruit from Brazil.

Efficacy of Pre- and Postinoculation Application of Fungicides to Expanding Young Citrus Leaves for Control of Melanose, Scab, and Alternaria Brown Spot.

In greenhouse trials, copper hydroxide, pyraclostrobin, and famoxadone were applied to actively growing young citrus seedlings to determine the duration of protection of young leaves provided by these fungicides against melanose, caused by Diaporthe citri, citrus scab, caused by Elsinoe fawcettii, and Alternaria brown spot, caused by Alternaria alternata. Fungicides were applied to different sets of potted plants of grapefruit for control of melanose, of rough lemon for control of scab, and of Dancy tangerine for control of Alternaria brown spot 1 to 6 days prior to inoculation, as well as on the day of inoculation. Leaf area of treated shoots was estimated on the day of fungicide application and the day of inoculation and disease severity evaluated subsequently. In most cases, copper hydroxide and famoxadone provided at least 50% control of all three diseases for only about 2 days after application. Generally, there was little or no disease control when the products were applied 4 or more days before inoculation. In contrast, pyraclostrobin usually provided a high level of control of all three diseases when applied up to 5 days prior to inoculation. The level of disease control decreased as the interval between a fungicide application and inoculation increased and the relationship between disease control and leaf expansion best fit a quadratic equation. Effective disease control was observed with copper hydroxide and famoxadone until leaf area had increased by 100 to 200%, whereas control with pyraclostrobin was observed up to 400 to 500% increase in leaf area. In postinoculation tests with scab and melanose, pyraclostrobin provided high levels of disease control (>75%) when applied up to 2 days after inoculation, whereas copper hydroxide and famoxadone had minimal postinoculation activity. Applications of pyraclostrobin to the spring flush growth of citrus trees are much more likely to provide control of melanose, scab, and Alternaria brown spot than those of famoxadone or copper hydroxide.

Comparison of Molecular Procedures for Detection and Identification of Guignardia citricarpa and G. mangiferae.

Citrus black spot, caused by Guignardia citricarpa, is a serious fruit spot disease and is widely distributed in Asia, southern Africa, and South America, but does not occur in North America or the Mediterranean region. A nonpathogenic species, G. mangiferae, is cosmopolitan with a wide host range and can colonize citrus fruit and leaves saprophytically. Detection and identification of Guignardia spp. on citrus fruit is necessary for epidemiological, management, and regulatory purposes. In this study, we compared published and unpublished polymerase chain reaction primer sets for their specificity and sensitivity in the detection and differentiation of the two Guignardia spp. All primers evaluated successfully identified the two species using purified DNA from fungal cultures or mycelia as source materials. However, some primer sets were not highly effective in detecting G. citricarpa when DNA was extracted directly from single characteristic black spot lesions on fruit. Thus, new primer pairs for both species were designed from the internal transcribed spacer region that were highly sensitive and specific for detection of G. citricarpa using DNA recovered from single lesions on fruit by a rapid DNA extraction procedure.

Saprophytic Colonization of Citrus Twigs by Diaporthe citri and Factors Affecting Pycnidial Production and Conidial Survival.

Melanose, caused by Diaporthe citri, produces reddish brown lesions on the fruit, leaves, and twigs of citrus trees, and greatly reduces the marketability of fresh fruit. Most of the inoculum is produced in pycnidia on dead twigs in the tree canopy, which exude large numbers of conidia in slimy masses. In this study, detached twigs inoculated with conidia were readily colonized and produced large numbers of pycnidia within 30 to 40 days when they were soaked 3 to 4 h on alternate days. Conidial production was measured by wetting twigs in a rain tower periodically and collecting the conidia in the runoff water. Production began after 80 days and continued for nearly 300 days. In other experiments, production of mature pycnidia on detached twigs was greatest at 94 to 100% relative humidity (RH) and at 28°C. Low RH and temperature, however, favored survival of conidia in exuded masses on twigs. In the field, colonization of detached twigs by D. citri was high in rainy season, moderate in spring and early fall, and minimal in late fall and winter. Twig colonization was positively related to the number of rain days and average temperature, but not to total rainfall. In another experiment, inoculated twigs placed in the tree canopy developed pycnidia and then produced conidial masses for about 200 days. D. citri is a serious pathogen, but a weak parasite, that survives primarily by colonization and reproduction on dead twigs.

Effect of Fungicides and Storage Conditions on Postharvest Development of Citrus Black Spot and Survival of Guignardia citricarpa in Fruit Tissues.

Citrus black spot (CBS) is caused by Guignardia citricarpa, which incites lesions on citrus fruit and can induce fruit drop. Quiescent infections occur during the spring and summer, and symptoms appear at fruit maturity or after harvest. Thus, fruit from citrus areas affected by CBS represent a risk for introduction of this pathogen into new areas. The effects of preventive field fungicide programs, postharvest fungicide drenches, packinghouse fungicide applications, and storage temperatures on postharvest symptom development and viability of G. citricarpa in lesions were evaluated in five experiments on Murcott tangor, Valencia oranges, and lemons. Preventive field treatments and fruit storage at 8°C consistently reduced postharvest CBS development, whereas a postharvest fungicide drench or packinghouse treatment with fungicides had no effect on postharvest symptom development. In a separate experiment, postharvest appearance of symptoms was related to the percentage of fruit with symptoms at harvest. The preventive field fungicide program also consistently reduced the percentage of isolation of G. citricarpa from affected fruit, whereas storage temperature and packinghouse fungicide treatment gave variable results. The viability of the fungus declined with storage time of fruit after harvest, but G. citricarpa could still be readily isolated regardless of treatment. In another experiment, the viability of the fungus in detached fruit or peel was minimally affected by temperature or moisture during storage. The frequency of successful isolation declined with time, but G. citricarpa was still recovered frequently from symptomatic tissue at later times. The most effective means to reduce postharvest development of symptoms is through preventive application of fungicides during the fruit growing season and storage of harvested fruit at cold temperatures. None of the measures evaluated substantially reduced viability of G. citricarpa, and the pathogen would likely be introduced on symptomatic fruit from citrus areas with CBS.

First Report of Alternaria Brown Spot of Citrus Caused by Alternaria alternata in Peru.

Alternaria brown spot, caused by Alternaria alternata (Fr.) Keissler, causes leaf, twig, and fruit lesions and reduces yield and fruit quality of many tangerines (Citrus reticulata Blanco) and their hybrids (3). In 2003, characteristic symptoms of brown spot were observed on young leaves and fruit of 'Minneola' tangelo in the Satipo Province of Peru. In 2004, the disease was discovered in the provinces of Chanchamayo, Leoncio Prado, and La Convención in the Junin, Huanuco, and Cusco regions, respectively, as well as in the Apurimac and the Ene valleys. In 2005, it was confirmed in the province of Oxapampa in the Pasco Region. Brown-to-black lesions surrounded by yellow halos and veinal necrosis were observed on young leaves, often causing abscission of young shoots and twig dieback. Light brown, circular lesions were observed on fruit, and when severe, resulted in premature abscission. Isolations from infected leaves and twigs were made on potato dextrose agar (PDA) with 10 µg/ml of benomyl. Colonies that developed after 5 days at 27°C were olive brown-to-black and produced small, muriform, pigmented conidia typical of A. alternata. On PDA without benomyl, gray colonies with conidia typical of Colletotrichum gloeosporioides were recovered frequently. Inoculation of three detached young shoots of 'Minneola' by spraying with a suspension of 105 conidia/ml of A. alternata produced leaf and twig symptoms characteristic of the disease after 48 h and confirmed pathogenicity of three isolates. Symptoms were not observed on control leaves sprayed with water nor on an equal number of leaves inoculated with a suspension of 105 conidia/ml of C. gloeosporioides. Reisolation of A. alternata from diseased tissue fulfilled Koch's postulates. DNA was extracted from 17 isolates and a partial endopolygalacturonase gene was amplified and sequenced (2). Sequences of all 17 isolates were identical, and in BLAST searches of the NCBI database, the closest matches were A. alternata accession nos. AY295023.1, AY295022.1, and AY295021.1 with 100, 99.8, and 99.8% sequence similarity, respectively. Phylogenetic analyses revealed that all isolates from Peru clustered with brown spot isolates from Israel, Turkey, South Africa, and Australia (1). These results, along with morphological characterization and pathogenicity tests, confirm the identity of the fungus as the tangerine pathotype of A. alternata. The disease has significantly reduced yield and the commercial value of fruit and may be a limiting factor for the production of susceptible cultivars in those areas of Peru. References: (1) T. L. Peever et al. Phytopathology 92:794, 2002. (2) T. L. Peever et al. Mycologia 96:119, 2004, (3) L.W. Timmer et al. Pages 19-21 in: Compendium of Citrus Diseases. 2nd ed. L. W. Timmer et al eds. The American Phytopathological Society, St. Paul, MN, 2000.

Effect of Lesion Age, Humidity, and Fungicide Application on Sporulation of Alternaria alternata, the Cause of Brown Spot of Tangerine.

Alternaria brown spot, caused by Alternaria alternata, causes yield losses and fruit blemishes on many tangerines and their hybrids in most citrus areas of the world where susceptible cultivars are grown. Although the conditions affecting infection and disease severity are known, little information is available on inoculum production on infected tissue. We found that sporulation on leaves began about 10 days after symptoms developed, was abundant from 20 to 40 days, and declined thereafter. Conidial production was far greater on leaf than on fruit or twig lesions. Spore production per unit area of leaf lesion was greater on the more susceptible hybrids, Minneola and Orlando tangelos, than on the less susceptible Murcott tangor. At 74% relative humidity, conidial production on leaf lesions was low, but it was abundant at 85, 92.5, 96, and 100%. Application of QoI or copper fungicides, but not ferbam, suppressed sporulation on leaf lesions for about 14 to 21 days after application. Additional applications did not appear to be more effective than a single spray in reducing inoculum production.

Resistance of Strawberry Cultivars to Crown Rot Caused by Colletotrichum gloeosporioides Isolates from Florida Is Nonspecific.

Isolates of Colletotrichum gloeosporioides from strawberry (Fragaria × ananassa) and native grape were tested for virulence on strawberry cultivars in field experiments for three seasons. Isolate aggressiveness and cultivar resistance were determined by the proportion of plants killed at a defined time. Each year, four to six isolates were inoculated on four to seven different cultivars, with a subset of isolates and cultivars evaluated again the next season. On the dates that disease was evaluated, incidence ranged from 10 to 84% for individual cultivars. Cultivar and isolate effects were significant in all three seasons, but there was no significant cultivar by isolate interaction in any season. Thus, resistance to C. gloeosporioides appears to be nonspecific. In the third season, one isolate of Colletotrichum fragariae from strawberry and one from oak were included. There was no significant cultivar by isolate interaction detected for this species, although there were significant differences among cultivars and isolates. When the resistance of cultivars to both species was compared, the rankings of cultivars were similar, but a modest cultivar by species interaction was evident. The cultivar Treasure was more resistant to crown rot caused by either species than any other cultivar tested.

Relationship of the Severity of Citrus Greasy Spot, Caused by Mycosphaerella citri, to Ascospore Dose, Epiphytic Growth, Leaf Age, and Fungicide Timing.

Greasy spot, caused by Mycosphaerella citri, produces leaf and fruit lesions and defoliates trees, resulting in reduced yields and fruit size. Techniques now available allow production of large numbers of ascospores and the quantification of epiphytic growth. The effects of ascospore dose, leaf age, and the timing of fenbuconazole sprays on epiphytic growth and disease severity was determined primarily on rough lemon seedlings in the greenhouse. Inoculation of leaves with 104 ascospores/ml resulted in rapid development of epiphytic growth and symptoms. At lower doses, epiphytic growth and symptoms developed more slowly and were less severe. There was a linear relationship between log10 of the ascospore dose and ratings of epiphytic growth and symptoms, and a linear relationship between the amount of epiphytic growth and symptom severity in greenhouse tests. On grapefruit trees treated with different fungicides in six field experiments, there also was a significant linear relationship between epiphytic growth of M. citri measured in August and symptom severity rated in February to March of the following year, but coefficients of determination were much lower than in greenhouse experiments. Leaf age from 10 to 60 days did not affect susceptibility of leaves to M. citri. Fenbuconazole applied up to 50 days prior to inoculation still reduced epiphytic growth and greasy spot severity under greenhouse conditions, but the postinoculation treatments were effective for only 30 days.

An Isolate of Alternaria alternata That Is Pathogenic to Both Tangerines and Rough Lemon and Produces Two Host-Selective Toxins, ACT- and ACR-Toxins.

ABSTRACT Two different pathotypes of Alternaria alternata cause Alternaria brown spot of tangerines and Alternaria leaf spot of rough lemon. The former produces the host-selective ACT-toxin and the latter produces ACR-toxin. Both pathogens induce similar symptoms on leaves or young fruits of their respective hosts, but the host ranges of these pathogens are distinct and one pathogen can be easily distinguished from another by comparing host ranges. We isolated strain BC3-5-1-OS2A from a leaf spot on rough lemon in Florida, and this isolate is pathogenic on both cv. Iyokan tangor and rough lemon and also produces both ACT-toxin and ACR-toxin. Isolate BC3-5-1-OS2A carries both genomic regions, one of which was known only to be present in ACT-toxin producers and the other was known to exist only in ACR-toxin producers. Each of the genomic regions is present on distinct small chromosomes, one of 1.05 Mb and the other of 2.0 Mb. Alternaria species have no known sexual or parasexual cycle in nature and populations of A. alternata on citrus are clonal. Therefore, the ability to produce both toxins was not likely acquired through meiotic or mitotic recombination. We hypothesize that a dispensable chromosome carrying the gene cluster controlling biosynthesis of one of the host-selective toxins was transferred horizontally and rearranged by duplication or translocation in another isolate of the fungus carrying genes for biosynthesis of the other host-selective toxin.

Citrus Black Rot is Caused by Phylogenetically Distinct Lineages of Alternaria alternata.

ABSTRACT Phylogenetic analysis revealed that isolates of Alternaria alternata causing black rot of citrus were associated with six well-supported evolutionary lineages. Isolates recovered from brown spot lesions on Minneola tangelo, leaf spot lesions on rough lemon, and healthy citrus tissue and noncitrus hosts were related closely to isolates from black-rotted fruit. Phylogenies estimated independently from DNA sequence data from an endopolygalacturonase gene (endoPG) and two anonymous regions of the genome (OPA1-3 and OPA2-1) had similar topologies, and phylogenetic analysis was performed on the combined data set. In the combined phylogeny, isolates from diverse ecological niches on citrus and noncitrus hosts were distributed in eight clades. Isolates from all lineages, regardless of ecological or host association, caused black rot in fruit inoculation assays, demonstrating that small-spored Alternaria isolates associated with different ecological niches on citrus and other plant hosts are potential black rot pathogens. These data also indicated that the fungi associated with black-rotted fruit do not form a natural evolutionary group distinct from other Alternaria pathogens and saprophytes associated with citrus. The use of the name A. citri to describe fungi associated with citrus black rot is not justified and it is proposed that citrus black rot fungi be referred to as A. alternata.

Baseline Sensitivities of Fungal Pathogens of Fruit and Foliage of Citrus to Azoxystrobin, Pyraclostrobin, and Fenbuconazole.

The baseline sensitivities for mycelial growth of foliar fungal pathogens of citrus, Colletotrichum acutatum, Alternaria alternata, Elsinoe fawcettii, Diaporthe citri, and Mycosphaerella citri, the causal agents of postbloom fruit drop, brown spot of tangerine, citrus scab, melanose, and greasy spot, respectively, were determined in vitro for azoxystrobin, pyraclostrobin, and fenbuconazole. The effective dose to reduce growth by 50% (ED50 values) was determined for each pathogen-fungicide combination using five isolates from different citrus areas of Florida and eight fungicide concentrations. A discriminatory dose for each combination was selected near the ED50, and the range of sensitivity of 50 to 62 isolates of each fungal species was determined. The effect of salicylhydroxamic acid (SHAM) on the sensitivity of the five fungal species to azoxystrobin and pyraclostrobin was determined. Since mycelial growth of A. alternata was insensitive to azoxystrobin, the effect of that fungicide with and without SHAM on spore germination was assessed. The ED50 values for most fungal pathogens of citrus were relatively high compared with foliar pathogens of other tree crops. Values for azoxystrobin ranged from a low of 0.06 µg/ml with E. fawcettii to a high of >100 µg/ml with A. alternata. With pyraclostrobin, the values ranged from a low of 0.019 µg/ml with D. citri to a high of 0.87 µg/ml with A. alternata. With fenbuconazole, the lowest ED50 value was 0.21 µg/ml with M. citri and the highest was 1.01 µg/ml with C. acutatum, but A. alternata and D. citri were not tested. SHAM was inhibitory to all species and reduced growth of D. citri greatly. Inclusion of SHAM in the medium did not greatly affect the sensitivity of mycelial growth of these fungi to azoxystrobin or pyraclostrobin, nor did it affect the ED50 values for conidial germination of A. alternata. The coefficients of variation for the sensitivity of 50 to 62 isolates of each species to these fungi ranged from 7.3% with the pyraclostrobin-C. acutatum combination to a high of 55.0% with the fenbuconazole- M. citri combination. Discriminatory doses have been established for these pathogen- fungicide combinations that should be useful for detecting major shifts in fungicide sensitivity.

Ascospore Deposition and Epiphytic Growth in Relation to Fungicide Timing for Control of Greasy Spot Rind Blotch Caused by Mycosphaerella citri.

Greasy spot rind blotch is a serious problem in Florida for the production of grapefruit (Citrus paradisi) for the fresh market. In the 1970s to the early 1980s, the disease was described in detail and the cause was determined to be Mycosphaerella citri, the same species responsible for greasy spot of foliage. The most appropriate timing for fungicide sprays was determined at that time, but peak ascospore release has changed in recent years. In the present study, the relationship of ascospore deposition and fungal growth on fruit was determined in order to more accurately time fungicide applications. Infection of fruit appears to occur similarly to that of leaves: by deposition of ascospores and germination to produce epiphytic growth followed by penetration of the fungus through stomata. Ascospore deposition occurred mostly in May and June, but epiphytic growth began only after the onset of the summer rainy season in June in 2002 and 2003. Ascospore deposition was lower in 2002 than in 2003, but development of epiphytic growth was similar in both years. Timing of fenbuconazole sprays was evaluated in the 2001, 2002, and 2003 seasons. Of the single-spray applications, those in July were the most effective, sprays in June and August were moderately effective, and those made in May or September were ineffective. Two- and three-spray programs from June through August were usually more effective than single sprays, and four monthly sprays from May to August were needed for a high level of control. Fungicide applications are needed about every 3 to 4 weeks after the beginning of the rainy season in June through August for a high level of control of rind blotch.

A gene with domains related to transcription regulation is required for pathogenicity in Colletotrichum acutatum causing Key lime anthracnose.

SUMMARY Colletotrichum acutatum causes Key lime anthracnose (KLA) and postbloom fruit drop (PFD) of citrus. We utilized restriction enzyme-mediated integration (REMI) mutagenesis to produce six non-pathogenic mutants from a KLA isolate after screening 1064 transformants on detached Key lime leaves. Subsequently, a gene designated KLAP1 (Key Lime Anthracnose Pathogenicity) was identified from one of the mutants and was demonstrated genetically to be required for pathogenicity to Key lime leaves. The predicted polypeptide encoded by KLAP1 contains a cAMP and cGMP-dependent protein kinase phosphorylation site, and two RGD (Arg-Gly-Asp) cell attachment sequences, a bipartite nuclear targeting sequence, a fungal G-protein alpha subunit signature, a putative metal-binding zinc finger (Cys(2)His(2)) and a putative HMG-I/Y ('high mobility group' non-histone chromatin protein encoding genes) DNA-binding domain (A+T hook), suggesting that KLAP1 may function as a transcription activator in C. acutatum. Sequences homologous to KLAP1 were detected in most C. acutatum isolates examined, and similarity was found in several classes of fungi, animals, plants and bacteria, indicating that KLAP1 is a putative, uncharacterized, conserved transcription activator in fungi. Targeted gene disruption of KLAP1 yielded mutants that were blocked in the penetration stage and were completely defective in pathogenicity on Key lime leaves, but remained pathogenic to flower petals. Complementation of a klap1-null mutant with a full-length KLAP1 gene clone restored complete ability to incite lesions on Key lime. The results indicate that KLAP1 is an important pathogenicity factor in C. acutatum.