Black spot, caused by Guignardia citricarpa, is not known in the U.S., but could be introduced on commercial shipments of fruit or on citrus material introduced by visitors. The other problem is that various spots or wounds on fruit in Florida may be colonized by G. mangiferae, a non-pathogenicspecies and confused with black spot. Thus, methods are needed to rapidly identify these fungi.
The major focus of our program is to evaluate the risk of introduction of black spot into the U.S. and means to reduce those risks. Primers specific to G. citricarpa and G. mangiferae have been obtained from other investigators in the U.S., Brazil, and South Africa, and new specific primers have been developed. All primers from other investigators efficiently detected G. citricarpa and were able to differentiate it from G. mangiferae using purified DNA or mycelium as source material. However, those primers were not highly effective for detection with DNA extracted from a single lesion on fruit. We designed new primers and found a single DNA extraction procedure that successfully detected G. citricarpa in single fruit lesions (Peres, et al., Plant Disease, accepted).
Symptoms of black spot can develop after harvest in transport or storage. In our studies, we found that intense fungicide application in spring and summer was the most effective means of preventing postharvest black spot symptoms. Fungicide applications immediately prior to or after harvest were ineffective in preventing postharvest symptom development. Cold storage delayed but did not prevent symptom development. G. citricarpa survived in whole fruit or detached peel regardless of the conditions of temperature or moisture under which they were held. The pathogen survived until tissues were decayed by other organisms.
In studies in Brazil where fruit was bagged to protect it from infection all except one-week periods, we found that infection occurred throughout the period from November to March. Ascospore peaks occurred following rain events and infection was strongly related to total rainfall, but not to average temperature (Fitopatol.Bras. 30:630-639).
All research on black spot has been conducted in Brazil and Argentina.
Citrus black spot, caused by Guignardia citricarpa, is a serious disease fruit spot disease and is widely distributed in Asia, southern Africa, and South America, but does not occur in North America or the Mediterranean. A non-pathogenic species, G. mangiferae, is cosmopolitan on a wide host range and can colonize citrus fruit and leaves.
For regulatory purposes, it is necessary to detect and identify Guignardia spp. that occur on citrus fruit. In this study, published procedures as well as some unpublished primer sets were compared for accuracy and sensitivity for detection and differentiation of the two species. All procedures successfully identified the two species using purified DNA or mycelium from culture as source materials. However, some primer sets were more effective than others in detecting G. citricarpa using DNA extracted from single lesions on fruit and none proved highly sensitive.
Thus, new primer pairs were designed from the ITS region that were specific for each of the two species. One primer pair was highly sensitive and specific for detection of G. citricarpa using DNA recovered by a rapid DNA extraction procedure. The pair specific for G. mangiferae effectively detected that species from decomposing leaf litter. Most of the published systems are effective for detection and differentiation of these two species of fungi, but the new procedure described herein specific, highly sensitive and easy to perform.
