Much of our current effort emphasizes the biology of Mycosphaerella citri and the development and maturation of pseudothecia; production of ascospores; and development of epiphytic mycelium prior to infection. In laboratory studies, we have developed an in vitro system for mating of Mycosphaerella citri. Matings of single-ascospore cultures indicate that M. citri is heterothallic and bipolar. Cytological studies indicate that pseudothecial ontogeny is typical of other loculoascomycetes.
In the field, the two mating types occur in a 1:1 ratio suggesting that mating is random (Phytopathology 94: 978-982). Wetting of detached infected leaves 3-4 times per week for 30-120 minutes each time brings about maximal production of pseudothecia. Longer wetting durations produce pseudothecia more quickly, but shorter ones maximize production. If leaves are wet continuously, they are colonized by saprophytes and may produce pseudothecial initials but never mature ascospores. The optimum temperature for pseudothecial production is 28°C with production slowing considerably at lower temperatures and dropping off rapidly at higher temperatures (Phytopathology 92: 1267-1275).
In other research, we investigated the effect of environmental factors on ascospore release with Tim Gottwald (USDA, Ft. Pierce) using a computer-controlled environmental chamber. Wetting of leaves by rainfall or irrigation triggers release of ascospores within 20-30 minutes. High humidity can also cause release of small numbers of spores. Once the pseudothecia are mature, many of the ascospores are released after the first wetting period with smaller numbers in the next 3-4 wetting periods. Subsequently, that leaf ceases to produce spores. In the field, ascospores are easily dispersed 100 m horizontally and 10 m vertically (Phytopathology 93: 1031-1036).
We are investigating treatments to speed decomposition of leaf debris, and thus, reduce spore production. Application of a 5% urea solution to detached infected leaves reduces pseudothecial development and ascospore production, and slows leaf decomposition. Calcium carbonate accelerates pseudothecial production, but reduces numbers of pseudothecia and ascospores. In the field, both lime and dolomitic limestone reduce production of pseudothecia and total ascospores by encouraging decomposition. Excess wetting and inoculation of leaves with saprophytic fungi may also be helpful in reducing spore production (Plant Dis. 87: 478-483).
Under grove conditions, the epiphytic mycelium begins to develop on spring flush leaves in May and June. It reaches a high level by the end of July, and those high levels are maintained but do not increase from August through December. A single application of fenbuconazole (Enable) in May or applications (June and July) to spring flush leaves controlled growth of epiphytic mycelium for many months. Even 18 months after leaf formation, greasy spot incidence, severity, and defoliation were minimal. An August application of Enable reduced epiphytic growth and delayed symptom production, but was much less effective than earlier sprays. A single spray in late July effectively controlled greasy spot on the summer flush (Plant Dis. 87: l186-192).
The relationship of ascospore deposition and fungal growth on fruit was determined to more accurately time fungicide applications. Infection of fruit appears to occur following deposition of ascospores and germination to produce epiphytic growth with subsequent penetration of the fungus through stomata.
Timing of fenbuconazole sprays was evaluated and, of the single-spray applications, July sprays were the most effective, June and August sprays 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 (Plant Dis. 89: 739-741).
We are currently investigating the effect of ascospore dose, leaf age, and fungicide timing in relation to inoculation time. Symptoms develop much more slowly when ascospore dose in low and leaves of all ages are susceptible. Fungicides applied before inoculation are effective for up to 50 days, but those made after applications are effective for 20-30 days.
We have determined the baseline sensitivity of M. citri in vitro to the fungicides azoxystrobin and pyraclostrobin and fenbuconazole. This information will be useful in evaluating the development of fungicide resistance in this pathogen. We are also continuing research on cultural control of greasy spot using products that reduce inoculum in leaf litter.
