Guignardia citricarpa , the causal agent of citrus black spot, forms airborne ascospores on decomposing citrus leaves and water‐spread conidia on fruits, leaves and twigs. The spatial pattern of diseased fruit in citrus tree canopies was used to assess the importance of ascospores and conidia in citrus black spot epidemics in São Paulo State, Brazil. The aggregation of diseased fruit in the citrus tree canopy was quantified by the binomial dispersion index ( D ) and the binary form of Taylor's Power Law for 303 trees in six groves. D was significantly greater than 1 in 251 trees. The intercept of the regression line of Taylor's Power Law was significantly greater than 0 and the slope was not different from 1, implying that diseased fruit was aggregated in the canopy independent of disease incidence. Disease incidence ( p ) and severity ( S ) were assessed in 2875 citrus trees. The incidence‐severity relationship was described ( R ² = 88·7%) by the model ln( S ) = ln( a ) + b CLL( p ) where CLL = complementary log‐log transformation. The high severity at low incidence observed in many cases is also indicative of low distance spread of G. citricarpa spores. For the same level of disease incidence, some trees had most of the diseased fruit with many lesions and high disease severity, whereas other trees had most of the fruit with few lesions and low disease severity. Aggregation of diseased fruit in the trees suggests that splash‐dispersed conidia have an important role in increasing the disease in citrus trees in Brazil.

Guignardia citricarpa is the causal agent of Citrus Black Spot (CBS), an important disease in Citriculture. Due to the expressive value of this activity worldwide, especially in Brazil, understanding more about the functioning of this fungus is of utmost relevance, making possible the elucidation of its infection mechanisms, and providing tools to control CBS. This work describes for the first time an efficient and successful methodology for genetic transformation of G. citricarpa mycelia, which generated transformants expressing the gene encoding for the gfp (green fluorescent protein) and also their interaction with citrus plant. Mycelia of G. citricarpa were transformed via Agrobacterium tumefaciens, which carried the plasmid pFAT-gfp, contains the genes for hygromycin resistance (hph) as well as gfp. The optimization of the agrotransformation protocol was performed testing different conditions (type of membrane; inductor agent concentration [acetosyringone - AS] and cocultivation time). Results demonstrated that the best condition occurred with the utilization of cellulose's ester membrane; 200 μM of AS and 96 h as cocultivation time. High mitotic stability (82 %) was displayed by transformants using Polymerase Chain Reaction (PCR) technique to confirm the hph gene insertion. In addition, the presence of gfp was observed inside mycelia by epifluorescence optical microscopy. This technique easy visualization of the behaviour of the pathogen interacting with the plant for the first time, allowing future studies on the pathogenesis of this fungus. The establishment of a transformation method for G. citricarpa opens a range of possibilities and facilitates the study of insertional mutagenesis and genetic knockouts, in order to identify the most important genes involved in the pathogenesis mechanisms and plant-pathogen interaction.

Citrus black spot (CBS), caused by Guignardia citricarpa, is the most important fungal disease of orange trees in Brazil. The spatial pattern of CBS-symptomatic trees was evaluated using the binomial dispersion index (D), Ripley's K function (K), and a Monte Carlo test for minimum mean distance (d) to understand the distribution of the pathogen. Disease was monitored in 7,790 citrus trees from four commercial groves. In one grove, disease incidence was assessed from 1999 to 2001 and, in the others, disease assessments were conducted only in 2002. Infected trees were aggregated based on the three statistical analyses used (D, K, and d) regardless of the CBS incidence. The binomial index of dispersion (D) indicated aggregation of CBS-affected trees for all groves and for various quadrat sizes (2 by 2, 3 by 3, 4 by 4… up to 10 by 10). According to Ripley's K function, the dependence among symptomatic trees comprised two to three neighboring trees. Disease dispersion occurred at distances below 24.7 m according to the test for d. This suggests that the dispersion of inoculum is highly important over short distances. As a consequence, the required sample size to achieve a level of accuracy of C = 20% increases exponentially with the decrease in incidence of CBS below 15% infected plants.