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

Abstract

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.

Genetic variation among Phyllosticta strains isolated from citrus in Florida that are pathogenic or nonpathogenic to citrus

“…P. citricarpa does not exhibit genetic variation in Florida and exhibits limited variation worldwide, based on three or four conserved genes. The lack of sequence variation supports the hypothesis that this pathogen has only recently been introduced to citrus-producing regions in the United States, Brazil, Africa, and Australia (Glienke et al, 2011;Er et al, 2013a;Schubert et al, 2012). In Asia (the center of origin of Citrus species), the diversity of P. citricarpa was greater when more Citrus species were sampled .…”

Endophytic Fungi of Citrus Plants

“…Particularly, the pathogenic P. citriasiana from south-east Asia [25], P. citrichinaensis from China [26], P. citrimaxima from Thailand [24], and P. paracitricarpa from Greece [27], and the non-pathogenic endophytic P. citribraziliensis from Brazil [28] and P. paracapitalensis from New Zealand, Italy, and Spain [27]. The isolation by the latter research group of P. citricarpa from specimen collected in citrus groves in Italy, Malta, and Portugal, following analogue findings in Florida [19,29], is expected to provide impulse for a more thorough assessment of distribution and pathogenicity of this species [30]. A very recent investigation carried out in Australia on several Citrus spp.…”

Phyllosticta citricarpa and sister species of global importance to Citrus

“…cultivars, with some lemons, limes, mandarins and late-maturing sweet oranges being the most susceptible (Kiely, 1948a(Kiely, , 1948b(Kiely, , 1949Kotzé, 1981Kotzé, , 2000Snowdon, 1990). CBS disease was reported for the first time in Australia (Benson, 1895), and is currently present in warm, summer rainfall areas of Asia, Africa, South America and North America (Kotzé, 1981;Schubert et al, 2012;Yonow et al, 2013).…”