Guignardia citricarpa causes citrus black spot (CBS) disease which was recorded, for the first time, in New South Wales, Australia in 1895 by Benson CITATION Hal08 \l 1033 (Halueendo, 2008). It caused heavy loss of citrus crop, particularly in coastal areas of New South Wales and Queensland. After about 30 years, CBS was seen in South AfricaCITATION JMK81 \l 1033 (Kotze, 1981). According to an extension paper from CREC/IFAS, University of Florida, it was first found in South Florida in March 2010 within 15 miles area around the South Florida near Immokalee, as center. By the first week of May on the same year, the disease was found in another location about 14 miles northeast from the original find.
Around the world, black spot can be found in Argentina, Australia, Brazil, China, Ghana, Mozambique, Philippines, South Africa, Sub-Saharan Africa, Taiwan and other regions of South America.
The sexual stage of the pathogen was discovered by Kiely in 1948 in New South Wales and he named it Guignardia citricarpa Kiely. The imperfect or asexual stage of the causal organism was known as Phoma citricarpa McAlp, but was later renamed to Phyllostictina citricarpa (McAlp) Petrak. However the name changed to Phyllosticta citricarpa in 1973 when the conidial stage was renamedCITATION JMK81 \l 1033 (Kotze, 1981).
Kingdom : Fungi
Phylum : Ascomycota
Class : Dothideomycetes
Subclass : Incertae sedis
Order : Botryosphaeriales
Family : Botryosphaeriaceaea
Genus : Guignardia
Species : G. citricarpa
Phoma citricarpa McAlpine, (1899)
Phoma citricarpa var. mikan Hara
Phyllosticta citricarpa (McAlpine) Aa, (1973)
Phyllostictina citricarpa (McAlpine) Petr., (1953)
Guignardia citricarpa is known to infect citrus and its hybrids. Among the commercial citrus varieties, Citrus limonia, C. nobilis, C. poonensis, C. tankan, grapefruits (C. paradisi), lemons (C. limon), limes (C. aurantifolia), mandarins (C. reticulata), oranges (C. sinensis) are the most susceptible.
Figure SEQ Figure \* ARABIC 1 ascospores of Guignardia citricarpa
The fungi are ascomycetes which are prevalent in the infected fallen leaves on the orchard. Under an electronic microscope, the ascospores are aseptate, elongated, spindle shaped with a slight bulge at the centre (fig 1).
Figure SEQ Figure \* ARABIC 2 cultured fungi of CBSWhen cultured in lab, colonies show greenish black masses of hyphal growth with mass of black pycnidia. Pycnidia are aggregated, superficial, globose to ampulliform, exuding a colourless, opaque conidial mass on their tips. The pycnidia are up to 250 μm diameter and pycnidial wall consist of several layers, 20–50 μm thick. Conidiophores produced on pycnidia are subcylindrical, branched from a supporting cell, coated in mucoid layer. Conidia are found singly, hyaline, aseptate, thin and smooth-walled, multiguttulate, ellipsoid to obovoid, tapering toward a narrowly truncate base, enclosed in a thin mucilaginous sheath, 1(–2) μm thick, and bearing a hyaline, mucoid apical appendage, 5–10(–17) × 1–1.5 μm, not branched, tapering towards an acute apexCITATION Gli11 \l 1033 (Glienke, et al., 2011).
Figure SEQ Figure \* ARABIC 3 (a) pycnidia forming on oatmeal agar; (b) conidiophores giving rise to conidia; (c) conidia
Biology of G. citricarpa
Though the asexual state of the citrus black spot fungus is documented to be Phyllosticta, another asexual structure has been recorded and named, Leptodothiorella which is referred to in the literature as the 'spermagonial'. Till the date, the exact function of the spermagonial state has not been found. The Phyllosticta state are found on fruit lesions, leaf lesions, dead twigs, fruit stalks and in abundance on leaves on the orchard floor, where as the Leptodothiorella state usually appears on fallen leaves before the development of ascocarps. The role of the asexual spore or conidia is unclear though it is supposed to have the function of secondary inoculation. Ascocarps occur throughout the year on leaf litter lying on the orchard floor. Mature ascospores are released from the ascoma during rainfall or irrigation and some are carried on the wind currents. Infection requires warm moist conditions. Early symptoms of black spot are not evident for more than 6 months after fruit set. Symptom expression on mature fruit is hastened by rising temperatures, high light intensity and drought stress and tree vigor. Older trees generally develop more black spot than the younger trees. Adequate hours of wetness, temperatures, and inoculum must be present simultaneously for infection to occurCITATION USD101 \l 1033 (USDA, 2010).
Cultures of G. citricarpa grow well on agar media like half strength PDA and complete media. The optimal temperature for growth has been reported to be 24-27°C. Germination of conidia has been enhanced by the presence of citric acid solutions at concentrations of 0.1-0.5%CITATION CAB \l 1033 (CABI and EPPO).
Figure SEQ Figure \* ARABIC 4 life cycle of Guignardia citricarpa
Fruit symptoms develop late in the season making detection difficult. Also, if fruit is not handled properly cold storage and processing, symptoms may develop postharvest. Incipient fruit infections can develop more fully postharvest, especially once the fruit are removed from cold storage CITATION USD10 \l 1033 (USDA, 2010).
Symptomatic citrus fruits may commonly show four types of symptoms: hard spot or shot-hole, false melanose, freckle spot, virulent spot or spreading spot, lacy-spot type lesion and cracked spot. Temperature and fruit maturity affect the types of symptom that appears. Diagnosis of black spot is difficult unless typical hard-spot symptoms containing pycnidia of the fungus are present on the fruit.
Hard spots are the most typical and diagnostic pre-harvest symptom of CBS. It is characterized by circular depressed lesions. It usually occurs on the side of sun exposure where fruit start changing color. Lesions are small, circular, and sunken with light brown to grey-white center and dark brown margin. Pycnidia are usually present in these lesions CITATION JMK81 \l 1033 (Kotze, 1981).
Figure SEQ Figure \* ARABIC 5 Left: Hard spot; Right: Freckle spot
False melanose are developed mostly on green fruits and do not contain pycnidia. They are observed as numerous small, slightly raised lesions that can be tan to dark brown. The lesions can coalesce as the season progresses and do not contain pycnidia. False melanose symptoms can develop into hard spot-type symptoms at the end of the season CITATION USD10 \l 1033 (USDA, 2010).
Freckle spots are similar to hard spots but they appear after the color of fruits changes or after picking. It has small reddish irregularly shaped lesions and can develop into either virulent spot or hard spot. Freckle spots are the sign of heavy infection.
Virulent spot lesions are sunken and irregular in shape and occur on heavily infected, mature fruit toward the end of the season. In high humidity, large numbers of pycnidia may develop. Virulent spot may cause premature fruit drop and serious postharvest losses since the symptoms may extend into the fleshy part of the fruit.
Lacy spot-type lesions are superficial, small, and yellow with dark yellow to brown centers and no defined margins. These are considered to be a variation of false melanose and have only been reported from South America.
Cracked spots type lesions are superficial, slightly raised, variable in size, brown to black with cracked surface and irregular margins. Pycnidia are not present. These lesions have often been associated with rust mite damage. Hard spot lesions can eventually form in the center of the lesions.
Symptoms are occasional in leaves and stems and prevails when disease incidence is high and not much of the control measures taken into consideration. They are most commonly found on lemons, a very susceptible species. Typically the infection in leaves remains latent, with no symptom development until after the leaves die. Young lesions are small, reddish, and slightly raised. A yellow halo can be associated with the lesions. Older lesions are small, round, sunken necrotic spots with gray centers. The lesions are bordered with a dark brown ring CITATION Mag03 \l 1033 (Magarey & Borchert, 2003).
Non-Pathogenic strain of G. citricarpa
Internal Transcribed Spacer sequence has disclosed two phylogenetically distinct group of G. citicarpa which was also supported by amplified fragment length polymorphism analysis CITATION Baa02 \l 1033 (Baayen, et al., 2002). Non-pathogenic isolate, G. mangiferae, was distinguished by their faster growth, colonies with entire margins rather than lobed type and production of pycnidia and ascomata in culture. It occurs widely on many tropical and sub-tropical non-cultivated and crop plants, including citrus and its geographical on citrus is much wider than pathogenic strain CITATION Per07 \l 1033 (Peres, Harakava, Carroll, Adaskaveg, & Timmer, 2007).
Citrus Black Spot has a great economic importance in the citrus production areas of the world. It is primarily a pre-harvest fruit disease which affects the rind of citrus, producing unsightly lesions, but can also produces postharvest symptoms of red spots on the fruits. This reduces the marketability of the fruits. G. citricarpa is also of phytosanitary concern in some countries to minimize the risks of introduction of the pathogens in the place where they are not yet established. All commercial varieties of citrus are susceptible except sour oranges (Citrus aurantium L.) and its hybrids. This arise heavy threat to the citrus growers all around the world.