First Report of Guignardia citricarpa Associated with Citrus Black Spot on Sweet Orange (Citrus sinensis) in North America.

In March 2010, citrus black spot symptoms were observed on sweet orange trees in a grove near Immokalee, FL. Symptoms observed on fruit included hard spot, cracked spot, and early virulent spot. Hard spot lesions were up to 5 mm, depressed with a chocolate margin and a necrotic, tan center, often with black pycnidia (140 to 200 µm) present. Cracked spot lesions were large (15 mm), dark brown, with diffuse margins and raised cracks. In some cases, hard spots formed in the center of lesions. Early virulent spot lesions were small (up to 7 mm long), bright red, irregular, indented, and often with many pycnidia. In addition, small (2 to 3 mm), elliptical, reddish brown leaf lesions with depressed tan centers were observed on some trees with symptomatic fruit. Chlorotic halos appeared as they aged. Most leaves had single lesions, occasionally up to four per leaf. Tissue pieces from hard spots and early virulent spots were placed aseptically on potato dextrose agar (PDA), oatmeal agar, or carrot agar and incubated with 12 h of light and dark at 24°C. Cultures that grew colonies within a week were discarded. Fourteen single-spore cultures were obtained from the isolates that grew slower than the Guignardia mangiferae reference cultures, although pycnidia formed more rapidly in the G. mangiferae cultures (1). No sexual structures were observed. Cultures on half-PDA were black and cordlike with irregular margins with numerous pycnidia, often bearing white cirrhi after 14 days. Conidia (7.1 to 7.8 × 10.3 to 11.8 µm) were hyaline, aseptate, multiguttulate, ovoid with a flattened base surrounded by a hyaline matrix (0.4 to 0.6 µm) and a hyaline appendage on the rounded apex, corresponding to published descriptions of G. citricarpa (anomorph Phyllosticta citricarpa) (1). A yellow pigment was seen in oatmeal agar surrounding G. citricarpa, but not G. mangiferae colonies as previously reported (1,2). DNA was extracted from lesions and cultures and amplified with species-specific primers (2). DNA was also extracted from G. mangiferae and healthy citrus fruit. The G. citricarpa-specific primers produced a 300-bp band from fruit lesions and pure cultures. G. mangiferae-specific primers produced 290-bp bands with DNA from G. mangiferae cultures. The internally transcribed spacer (ITS) of the rRNA gene, translation-elongation factor (TEF), and actin gene regions were sequenced from G. citricarpa isolates and deposited in GenBank. These sequences had 100% homology with G. citricarpa ITS sequences from South Africa and Brazil, 100% homology with TEF, and 99% homology with actin of a Brazilian isolate. Pathogenicity tests with G. citricarpa were not done because the organism infects immature fruit and has an incubation period of at least 6 months (3). In addition, quarantine restrictions limit work with the organism outside a contained facility. To our knowledge, this is the first report of black spot in North America. The initial infested area was ~57 km2. The disease is of great importance to the Florida citrus industry because it causes serious blemishes and significant yield reduction, especially on the most commonly grown 'Valencia' sweet orange. Also, the presence of the disease in Florida may affect market access because G. citricarpa is considered a quarantine pathogen by the United States and internationally. References: (1) R. P. Baayen et al. Phytopathology 92:464, 2002. (2) N. A. Peres et al. Plant Dis. 91:525, 2007 (3) R. F. Reis et al. Fitopath Bras. 31:29, 2006.

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.

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 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.

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.

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.

Factors Affecting Pycnidium Production of Diaporthe citri on Detached Citrus Twigs.

Melanose, caused by Diaporthe citri, produces black-to-reddish brown lesions on twigs, leaves, and fruit of citrus and reduces the external quality of fruit destined for the fresh market. Inoculum for infection is produced primarily in pycnidia formed on dead twigs, and conidia are dispersed by rainwater. In laboratory studies, the effect of moisture, temperature, twig size, and melanose severity on pycnidium production on detached twigs was investigated. Pycnidium production was greatest when twigs were soaked for 3 to 4 h on alternate days three times per week and the temperature was 28°C. Production was greatest on twigs 3 to 5 mm in diameter and less on thinner or thicker twigs. Pycnidium production was related linearly to melanose severity on the twigs, and almost no pycnidia were produced on asymptomatic twigs. In the field, pycnidium production was greatest on detached, melanose-affected twigs placed in the canopy monthly during January to April than it was on twigs placed in the canopy during other months. The largest number of pycnidia was produced from May to August when fruit is most susceptible. The number of pycnidia produced was related significantly to degree-days above 20°C and weakly related to cumulative rainfall. Knowledge of inoculum production peaks may assist in timing of pruning and fungicide sprays.

Mating and Pseudothecial Development in Mycosphaerella citri, the Cause of Citrus Greasy Spot.

ABSTRACT Greasy spot, caused by Mycosphaerella citri, is a serious disease of citrus in the Caribbean basin. M. citri is a loculoascomycete and produces pseudothecia in decomposing leaves after intermittent wetting and drying. A new in vitro mating technique was developed for production of pseudothecia on sterilized leaf disks in petri dishes. Of the single-ascospore cultures that were recovered from individual asci, four were one mating type and four were a second mating type (tentatively designated mat+ and mat-), indicating that M. citri probably is heterothallic and bipolar like most other loculoascomycetes. Most populations of ascospores recovered from individual leaves or from leaves from groves of different citrus species and various locations had a 1:1 ratio of mating types consistent with random mating. Cytological studies demonstrated that the ontogeny of pseudothecial development was similar to other loculoascomycetes. The formation of mature pseudothecia required 30 to 45 cycles of wetting and drying of infected, dead leaves which required approximately 60 to 90 days. The in vitro system for pseudothecial production and the knowledge of the mating system in M. citri will facilitate genetic studies of this important pathogen.

Environmental Factors Affecting the Release and Dispersal of Ascospores of Mycosphaerella citri.

ABSTRACT Greasy spot, caused by Mycosphaerella citri, produces a leaf spot disease affecting all citrus species in Florida and the Caribbean Basin. M. citri produces pseudothecia and ascospores, which are considered the principal source of inoculum, in decomposing leaves on the grove floor. In studies using a computer-controlled environmental chamber, a single rain event triggered release of most mature ascospores beginning 30 to 60 min after the rain event. Additional rain events did not bring about further release. High relative humidity without rain triggered release of low numbers of ascospores, but vibration and red/infrared irradiation had little or no effect on ascospore release. After three to four cycles of wetting and drying of leaves, all pseudothecia had matured and released their ascospores. In the field, ascospores were detectable starting about 2 h after the beginning of a rain or irrigation and most ascospores were released within 16 h. Ascospore release was greatest following rain events and somewhat less following irrigations, and low numbers of ascospores were detectable on days without precipitation. Ascospore numbers declined linearly with horizontal distance from the source and as a function of the logarithm of ascospore numbers with vertical distance. Low numbers of ascospores were detected 7.5 m above the ground and 90 m downwind from the grove. Ascospore release can be advanced by irrigating frequently during dry, nonconducive conditions to stimulate ascospore release when environmental conditions are unfavorable for infection, but the eventual effects on disease severity are uncertain.

Relationship of Epiphytic Growth of Mycosphaerella citri to Greasy Spot Development on Citrus and to Disease Control with Fenbuconazole.

Greasy spot, caused by Mycosphaerella citri, produces foliar lesions and severe defoliation of citrus trees. Ascospore production and deposition by M. citri, development of epiphytic growth, and symptoms were monitored on grapefruit trees in the field and on rough lemon trap plants for 2 years. Ascospore production and deposition peaked in April to May both years. However, epiphytic mycelium did not develop extensively until the summer rainy season was well underway in July. Some epiphytic growth and symptoms formed on trap plants placed in the grove for 2-week periods throughout the year. In the summer, epiphytic growth was apparent 15 days after the exposure period, and symptoms appeared about 60 days after exposure, but development was much slower in cooler and drier months. One or two fenbuconazole applications before the development of epiphytic mycelium in July completely controlled greasy spot on spring growth leaves for 12 to 18 months. Applications in July or August were less effective. Epiphytic mycelium developed more rapidly on summer growth; therefore, fungicide applications need to be timed more precisely.

Effect of Urea, CaCO3, and Dolomite on Pseudothecial Development and Ascospore Production of Mycosphaerella citri.

Citrus greasy spot, caused by Mycosphaerella citri, produces lesions on leaves, followed by premature defoliation, and rind blotch on fruit. Ascospores produced in leaf litter represent the major source of inoculum. The effect of treatment of leaf litter with urea, CaCO3, or dolomite on the development of pseudothecia and ascospore production was evaluated. In laboratory experiments, one urea application reduced production of pseudothecia and ascospores by up to 90%, but did not affect time of production of pseudothecia or ascospores or rate of leaf decomposition. Two applications of urea delayed leaf decomposition. As the rates of CaCO3 or dolomite were increased, pseudothecial incidence, density, time to ascospore production, and total numbers of ascospores decreased and the rate of leaf compostion increased. Immature pseudothecia on leaves treated with urea or CaCO3 degenerated and produced fewer ascospores per pseudothecium. The results observed in microplot studies in the field were similar to those observed in laboratory experiments. The number of days to pseudothecia and ascospore production and the pseudothecial incidence and density were negatively related to the rate of CaCO3 or dolomite applied. Application of CaCO3 dolomite, or urea to leaf litter can reduce inoculum and be useful in an integrated program of greasy spot management.

Environmental Factors Affecting Pseudothecial Development and Ascospore Production of Mycosphaerella citri, the Cause of Citrus Greasy Spot.

ABSTRACT Mycosphaerella citri, the cause of citrus greasy spot, produces pseudothecia and ascospores in decomposing leaf litter on the grove floor. In laboratory studies, the effect of wetting and drying and temperature on the formation, maturation, and production of pseudothecia and ascospores was evaluated on mature, detached grapefruit leaves. Production of pseudothecia was most rapid when leaves were soaked five times per week for 2 h per day, but pseudothecial density and total ascospore production were greatest when leaves were soaked three times per week for 2 h per day. In duration of wetting studies, 3 h per day, 3 days per week brought about the most rapid production, but 10 to 30 min per day resulted in production of the most pseudothecia and ascospores. Pseudothecia and ascospore production were greatest at 28 degrees C and declined rapidly at lower and higher temperatures. Maturation of pseudothecia was slow at 20 and 24 degrees C, but production was high at 24 degrees C; at 32 degrees C, pseudothecia matured rapidly, but degenerated quickly. No mature pseudothecia were produced on leaves maintained continuously under wet conditions. In field studies, leaves were placed on the grove floor monthly from April 2000 to September 2001. Pseudothecia production was rapid during the summer rainy season from June to September. Pseudothecia produced on leaves placed in the grove from October to May developed and matured more slowly but were produced in much larger numbers than in summer. The number of days to first pseudothecial initials, 50% maturation, first discharge of ascospores, leaf decomposition, as well as pseudothecial density and incidence, were negatively related to average temperature. Total ascospore production was unrelated to temperature.

Carbon Loss and Germinability, Viability, and Virulence of Chlamydospores of Fusarium solani f. sp. phaseoli After Exposure to Soil at Different pH Levels, Temperatures, and Matric Potentials.

ABSTRACT (14)C-labeled chlamydospores of Fusarium solani f. sp. phaseoli were exposed to soil at 5, 15, 25, or 30 degrees C at pH 5 or 8 and water potential of -1 kPa or to soil at 0, -1, or -10 kPa at 25 degrees C at pH 6.9. Total carbon loss was greatest at 25 or 30 degrees C at pH 8 and -1 kPa. (14)CO(2) from respiration of chlamydospores and from soil microbes utilizing chlamydospore exudates accounted for the largest share of total carbon loss under all conditions. (14)(CO)(2) from soil microbial metabolism of (14)CO(2) exudates of chlamydospores was greatest in soil at 15, 25, and 30 degrees C, pH 8, and at either -1 or -10 kPa. Chlamydospore germinability in the absence of a C source (nutrient independence), viability in potato-dextrose broth, and virulence to kidney bean declined rapidly after exposure to soil at high temperatures (25 and 30 degrees C), pH 8, and the higher matric potentials (0 to-1 kPa). By contrast, germinability remained high (>50%), as did virulence, in soil at 5 degrees C and -10 kPa even after 70 days of incubation. Carbon loss was inversely correlated with germinability, viability, and virulence after exposure to soil at different pH levels, temperatures, and matric potentials.

Interchromosomal asynchrony of DNA replication in polytene chromosomes of Drosophila pseudoobscura.

Analysis of 3H-thymidine autoradiograms of late third instar larval salivary glands of Drosophila pseudoobscura revealed a unique example of asynchrony of replication in the autosome complement. The two autosomal arms, 2 and 3, show similar labeling pattern during the initial phases, DD to 3C, and thereafter, the chromosome 3 has fewer labeled sites than chromosome 2 until the most terminal pattern, 1D. Detailed sitewise analysis of 3H-thymidine labeling shows that while nearly 54% of the sites examined in chromosome 2 have a labeling frequency greater than 50%, only 13% of all sites in chromosome 3 have labeling frequency at that range. The number of labeled sites on chromosome 3 plotted against that on chromosome 2 shows a hyperbolic profile rather than a linear relationship. The silver grain ratio of the 2nd to 3rd increases from 1.5 to 3.1 through different stages of the cycle. These results suggest that both chromosomes start replication simultaneously but the third chromosome appears to complete the replication earlier than the second. These data open up the possibility of separate control mechanisms for the initiation and termination of DNA replication in polytene chromosomes.