Sensitivity of Colletotrichum acutatum Isolates from Citrus to Carbendazim, Difenoconazole, Tebuconazole, and Trifloxystrobin.

Postbloom fruit drop (PFD) of citrus is caused by the Colletotrichum acutatum and C. gloeosporioides species complexes. The disease is important when frequent rainfall occurs during the flowering period of citrus trees. In Brazil, until 2012, PFD was mainly controlled by preventive applications of the methyl-benzimidazole carbamate (MBC) carbendazim and demethylation-inhibitor (DMI) fungicides such as difenoconazole. Since then, mixtures containing the DMI tebuconazole and the quinone-outside inhibitor (QoI) trifloxystrobin have been commonly used. Fungicides are often applied preventively, sometimes even when conditions are not conducive for PFD development. Excessive fungicide applications may favor the selection of resistant populations of Colletotrichum spp. In this study, we assessed the fungicide sensitivity of C. acutatum isolates collected during the two distinct periods of PFD management in Brazil: before and after the trifloxystrobin and tebuconazole mixture became widely employed. The sensitivity of 254 C. acutatum isolates to carbendazim and difenoconazole and of 164 isolates to tebuconazole and trifloxystrobin was assessed. Mycelial growth inhibition of these isolates was evaluated for all the fungicides using either serial dilution of fungicide rates or the spiral gradient dilution method. In addition, inhibition of conidial germination was also assessed for trifloxystrobin. Analysis of partial ß-tub, cytb, and cyp51b gene sequences did not reveal any mutations related to resistance to MBCs, QoIs, and DMIs, respectively. In mycelial growth assays, mean EC50 values were 0.14, 0.11, and 0.21 µg/ml for difenoconazole, tebuconazole, and trifloxystrobin, respectively. The conidial germination inhibition by trifloxystrobin was similar among the tested isolates, and the mean EC50 value was 0.002 µg/ml. All isolates had similar mean mycelial growth inhibition for carbendazim, regardless of the fungicide concentrations. Therefore, based on similar EC50 values and molecular analyses, no shift in the sensitivity of isolates has been observed to the fungicides commonly used in different citrus-producing areas in Brazil.

Microscopic analysis of colonization of Colletotrichum abscissum in citrus tissues.

Postbloom fruit drop (PFD), caused mainly by Colletotrichum abscissum, is one of the most severe citrus diseases and can causes up to 80% fruit loss in favorable climatic conditions. According to the literature, other Colletotrichum species colonize hosts using distinct strategies: intracellular hemibiotrophic or subcuticular intramural necrotrophic colonization. However, so far, for C. abscissum only the necrotrophic stage has been described and some aspects remain unclear in PFD disease cycle. To better understand the disease cycle, microscopy studies could be applied. However, even using eGFP strains (expressing green fluorescent protein), the results are unclear due to the autofluorescence of citrus leaves. To eliminate this problem and to study the interaction between C. abscissum-citrus we used a destaining and staining methodologies, and we observed that in leaves, even applying injury before inoculation, C. abscissum does not colonize adjacent tissues. Apparently, in the leaves the fungus only uses the nutrients exposed in the artificial lesions for growth, and then produces large amount of spores. However, in flowers, C. abscissum penetrated and colonized the tissues of the petals 12 h after inoculation. In the early stages of infection, we observed the development of primary biotrophic hyphae, suggesting this species as a hemibiotrophic fungus, with a short biotrophic phase during flower colonization followed by dominant necrotrophic colonization. In conclusion, the use of an eGFP strain of C. abscissum and a different methodology of destaining and staining allowed a better understanding of the morphology and mechanisms used by this citrus pathogen to colonize the host.

Saccharomyces cerevisiae: A novel and efficient biological control agent for Colletotrichum acutatum during pre-harvest.

In this study, we evaluated the efficiency of six isolates of Saccharomyces cerevisiae in controlling Colletotrichum acutatum, the causal agent of postbloom fruit drop that occur in pre-harvest citrus. We analyzed the mechanisms of action involved in biological control such as: production of antifungal compounds, nutrient competition, detection of killer activity, and production of hydrolytic enzymes of the isolates of S. cerevisiae on C. acutatum and their efficiency in controlling postbloom fruit drop on detached citrus flowers. Our results showed that all six S. cerevisiae isolates produced antifungal compounds, competed for nutrients, inhibited pathogen germination, and produced killer activity and hydrolytic enzymes when in contact with the fungus wall. The isolates were able to control the disease when detached flowers were artificially inoculated, both preventively and curatively. In this work we identified a novel potential biological control agent for C. acutatum during pre-harvest. This is the first report of yeast efficiency for the biocontrol of postbloom fruit drop, which represents an important contribution to the field of biocontrol of diseases affecting citrus populations worldwide.