Review of Guignardia citricarpa Kiely, the causal agent of citrus black spot 

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CHAPTER 2 Review of Guignardia citricarpa Kiely, the causal agent of citrus black spot

The pathogen, Guignardia citricarpa

Origin and distribution of Guignardia citricarpa

Guignardia citricarpa Kiely originated collectively with its host, Citrus L., from South East Asia (Smith et al., 1997). The asexual form of the fungus was first described by McAlpine in 1899 as Phoma citricarpa McAlpine from symptomatic citrus fruit in Australia. Since then it had two name changes and Phyllosticta citricarpa (McAlpine) Aa is currently the accepted name (Van der Aa, 1973; Van der Aa & Vanev, 2002). The sexual form was described by Kiely (1948b) as G. citricarpa from citrus leaf litter in Australia. The spermatial state or synanamorph is a Leptodothiorella and the species has not been described (Van der Aa, 1973; Baayen et al., 2002).
Today, the citrus pathogen is widespread and occurs in Argentina, Australia, Bhutan, Brazil, China, Ghana, India, Indonesia, Kenya, Mozambique, Nigeria, Philippines, South Africa (SA), Swaziland, Taiwan, United States of America (USA), Uruguay, West Indies, Zambia and Zimbabwe (European Union, 1998; Baayen et al., 2002; Paul et al., 2005; Lemon & McNally, 2010; Schubert et al., 2010). G. citricarpa has not been recorded in Mediterranean and European countries, or in Chile, Japan and New Zealand (European Union, 1998; Baayen et al., 2002; Paul et al., 2005; Everett & Rees-George, 2006a).

Guignardia species on citrus

There are two main morphologically similar Guignardia species occurring on Citrus, G. citricarpa, causing black spot or symptomless infections in Citrus, and Guignardia mangiferae A.J. Roy, non-pathogenic to Citrus, causing only symptomless infections that remains latent (Meyer et al., 2001; Baayen et al., 2002; Bonants et al., 2003). The endophytic nature of the fungi on citrus caused confusion in the past, since all isolates of Guignardia obtained from Citrus was considered to be the citrus pathogen, G. citricarpa. The latent or endophytic nature of G. citricarpa was first recognised by Cobb (1897), and the pathogen has ubiquitously been isolated from healthy citrus tissue (McOnie, 1964a, d; Araújo et al., 2001; Glienke-Blanco et al., 2002; Bonants et al., 2003; Baldassari et al., 2008).
Both species of Guignardia may simultaneously colonise the same citrus tissue, being either symptomatic or symptomless leaves, twigs or fruit (McOnie, 1964a, d; Baayen et al., 2002; Bonants et al., 2003; Baldassari et al., 2006) and have been reported to coexist in a single black spot lesion (Baldassari et al., 2008). Furthermore both species have been reported from cultivars not susceptible to CBS, including Seville sour orange (Citrus aurantium L.) and Tahiti acid lime (Citrus latifolia Tan.) (McOnie, 1964d; Baldassari et al., 2008), contributing further to the uncertainty surrounding the identity of the pathogen for so many years.
Apart from pathogenicity, these species differ in culture characteristics and host range. Isolates of G. citricarpa can be distinguished from G. mangiferae by a combination of several characteristics (Table 2.1), although none of the characteristics on its own was found to separate both species unambiguously (Baayen et al., 2002). One of the more useful characteristics is the yellow pigment production at the edge of colonies on Oats agar (OA). Only isolates of G. citricarpa produce a yellow pigment on OA and it is reported to be a consistent trait in G. citricarpa isolates from various citrus materials (Baayen et al., 2002; Baldassari et al., 2008). However, Wulandari et al. (2009) reported three isolates of G. mangiferae producing yellow pigment on OA. Also, sporulation is required for confirmation as other fungi may resemble G. citricarpa while still sterile.
Another important characteristic is the production of spores in culture and although the feature is consistent in fresh isolates, there are numerous conflicting reports. Isolates from G. citricarpa never produces ascospores in culture, irrespective of what growth media are used, and infertile pseudothecia has been reported to occur rarely (McOnie, 1964b, d; Korf, 1998; Baayen et al., 2002; Baldassari et al., 2008). Isolates of G. mangiferae produces both pycnidiospores and ascospores in culture, although not all isolates formed fertile pseudothecia (Kiely, 1948b; Baayen et al., 2002; Baldassari et al., 2008). All reports on isolates of G. citricarpa producing ascospores in culture (Frean, 1964; Brodrick, 1969; Wager, 1952) are believed to be erroneous. Results of Lemir et al. (2000), who claimed to have produced pseudothecia of G. citricarpa in culture, could not be repeated (Baayen et al., 2002; Baldassari et al., 2008; M. Truter, unpublished data).
Molecular studies on Guignardia isolates from Citrus and other hosts indicated that G. citricarpa could clearly distinguished morphological similar isolates as a separate species (Meyer et al., 2001; Baayen et al., 2002; Wulandari et al., 2009). Meyer et al. (2001) used restriction enzyme digestion fingerprints of the polymerase chain reaction (PCR) product of a portion of the internal spacer region (ITS) to indicate the two species, while Baayen et al. (2002) used ITS sequence analysis and amplified fragment length polymorphic fingerprint patterns. These and other molecular studies on Guignardia isolates resulted in development of species-specific PCR primers that provided fast, accurate and reliable techniques to distinguish and detect the species without reservation (Meyer et al., 2001; Baayen et al., 2002; Bonants et al., 2003; Meyer et al., 2006; Everett Rees-George, 2006b; Peres et al., 2007; Van Gent-Pelzer et al., 2007; Stringari et al., 2009).
It has been suggested that a third Phyllosticta species is associated with Citrus, but only as symptomless infections (Van der Aa & Vanev, 2002; Baayen et al., 2002). Stringari et al. (2009) recently indicated that isolates from symptomless C. limon in Brazil belonged to Phyllosticta spinarum (Died.) Nag Raj & M. Morelet based on sequence data. Wulandari et al. (2009) also referred to one of these isolates from Brazil, and subported that it could be P. spinarum. Besides Possiede et al. (2009) referring to the same P. spinarum isolates on citrus as Stringari et al. (2009), no further record(s) of this fungus on citrus are known.
A fourth Phyllosticta species, Phyllosticta citriasiana Wulandari, Crous & Gruyter, has recently been described from pummelo, Citrus maxima Merr., causing citrus tan spot (Wulandari et al., 2009). The teleomorph was indicated as unknown. All isolates from the newly described species were obtained from spotted fruit of C. maxima from China, Thailand and Vietnam (Wulandari et al., 2009). Fruit symptoms are similar to those produced by G. citricarpa, consisting of shallow lesions with a small central grey to tan crater usually with a dark brown rim, 3-10 mm in diameter (Wulandari et al., 2009). P. citriasiana can be distinguished from G. mangiferae by having smaller conidia with a narrower mucoid sheath, and from P. citricarpa by having larger conidia, longer conidial appendages and not producing any diffuse yellow pigment when cultivated on OA (Wulandari et al., 2009). In culture, colonies of P. citriasiana are also darker shades of grey and black on OA, malt extract agar, potato-dextrose agar and cornmeal agar than observed in the other two species (Wulandari et al., 2009).

Morphology of Guignardia citricarpa

Pseudothecia are produced solitary (125-135 µm in diameter) or in groups of two (220-240 µm) and three (340-360 µm). Pseudothecial wall are 20-22 µm thick, carbonaceous dark brown by transmitted light and globose. Pseudothecia are sub-epidermal, finally erumpent, no stroma present nor distinct beak, but an ostiole of 14-16 µm in diameter are present at maturity. Paraphyses and periphyses are absent. Pseudothecia are produced on the ventral and dorsal surfaces of decaying citrus leaves, but have never been found on fruit (Kiely, 1948b; Van der Aa, 1973).
Asci (50-85 x 12-15 µm) are produced from the base of a pseudothecium, 45 to 60 in number, clavate; cylindrical, eight spored and uniseriate (Kiely, 1948b). Ascopsores are hyaline to granular grey, usually with one large central guttule at maturity. Ascospores are non-septate but occasionally with septum near one end of the spore, 8.0-17.5 x 3.3-8.0 µm with a small round clear gelatinous cap at each end (Kiely, 1948b).
Pycnidia are produced on citrus leaves, petioles, twigs and fruit (Van der Aa, 1973). Pycnidia are 70-330 µm in diameter, subhyaline to brownish on leaves, brown to almost black on fruit, globose or depressed on leaves, pyriform on fruit, flat or conspicuously papillate with a circular pore of 10-15 µm diameter. Stroma developed on fruit only, are subhyaline to dark brown and 5-18 µm in diameter. Conidiogenous cells are cylindrical and 4-8 x 2-3.5 µm. Under ideal conditions for their development, pycnidia are closely studded over the entire leaf surface. They can occur on either the dorsal or ventral surfaces of the leaf, but are usually thickest on the one side only, the side or portion of the leaf exposed to the sun’s radiation (Darnell-Smith, 1918; Kiely, 1948b).
Pycnidiospores still attached to the sporophore possess a terminal gelatinous cap, which later shrink to form the appendage, 5-15 µm in length. Pycnidiospores are one-celled, obovoidal, ellipsoidal or subglobose, somewhat clavate when young, with a truncate base, broadly rounded apically and slightly indented, 6-13 x 5-9 µm, usually 9-10 x 6-7 µm (Van der Aa, 1973). They may have one or two nuclei, generally two (Darnell-Smith, 1918). Pycnidiospores are usually hyaline with granular contents and sometimes having a greenish hue. More than one crop of pycnidiospores can be produced as the sporogenous layer is regenerative (Kiely, 1948b).
Spermatial state occurs both in pure culture and on the host and usually develops simultaneously with the conidial state, but is much more scarcely found (Van der Aa, 1973). Fruiting bodies are similar to those of the conidial state. Spermatiogenous cells are elongated cylindrical and 4-10 x 0.5-2 µm. Spermatia are dumb-bell shaped, seldom cylindrical, straight to slightly curved and 5-8 x 0.5-1 µm.
The mycelium exhibits much diversity. The extreme tips may be pointed or round, hyphae being thin, hyaline, and almost devoid of septa (Darnell-Smith, 1918). Older hyphae become thicker, septa more numerous and olive-green in colour. In the older hyphae, septa are numerous, dark greenish-brown in colour, and the contents of the cells granular. The cells may be oblong or round and often carry numerous short, round, protuberances. Hyphae anastomose readily with one another (Darnell-Smith, 1918).
Cultures of G. citricarpa on potato-dextrose agar are dark brown to black; mycelium is mostly submerged, thick and prostrate. Colonies are slow growing, reaching a diameter of 70 mm in 20 days on various media at 24°C (Van der Aa, 1973). Stromata develop within eight days as hard, black masses, resembling those on fruits, pyriform, globose or cylindrical, with one to numerous conidial and spermatial cavities in the upper region (Van der Aa, 1973).

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Sporulation

All attempts to promote pseudothecial development of G. citricarpa in vitro were unsuccessful (McOnie, 1964d; Korf, 1998; Baayen et al., 2002; Baldassari et al., 2008) and although Lemir et al. (2000) claim to have produced pseudothecia in culture, their results were never repeated. With our current knowledge about G. mangiferae, we can conclude that reports on in vitro ascospore production of G. citricarpa (Frean, 1964, 1966; Brodrick, 1969; Wager, 1952) are erroneous. Other methods for the production of pseudothecia on water agar medium augmented with leaf pieces were described, but for members of the genus Guignardia and not for G. citricarpa specifically (Petrini et al., 1991; Furukawa & Kishi, 2002).
Brodrick and Rabie (1970) investigated the effects of light and temperature on the sporulation on artificial culture medium. Incubation under continuous light resulted in significantly higher counts of pycnidiospores produced than under alternating light/dark or continuous dark. Incubation at 27°C resulted in significantly more pycnidiospores produced on flavedo pieces than at 20°C, whereas the reverse was true for pycnidiospore production on Potato Dextrose Agar. Numbers of pycnidiospores produced were significantly higher in all the treatments after 15 days than after 10 and 20 days. At 20 days, it was possible that the pycnidiospores remained embedded in the gelatinous matrix in the pycnidium and were not released under the conditions of the experiment.

 Spore germination

Since ascospores of G. citricarpa cannot be produced in vitro, very few studies have investigated the germination of ascospores. According to Kiely (1948b) ascospores take more than 24 h to germinate in vitro at 25°C and 4 days to reach 98% germination. In another study, germination was investigated in vitro and in plantae and germination of ascospores on lemon (Citrus limon (L.) Burn. f.) leaves varied from 14 to 91% after 24 h and most did not show an increase after 48 h compared to 24 h (McOnie, 1967).
In vitro germination of pycnidiospores of P. citricarpa has been reported to be very slow, with only a few spores germinating after several days (Darnell-Smith, 1918). Germination of pycnidiospores in tap water has been reported, albeit at varying degrees (Kiely, 1948b; Wager, 1952). Spore germination was stimulated by extracts of orange peel or citric acid solutions at concentrations of 0.1-0.5% (Darnell-Smith, 1918; Kiely, 1948b). Maximum germination of nearly 80% has been obtained using 0.3% citric acid solution and incubating spores for 4 days at 25°C in a damp chamber (Kiely, 1948b). Freshly exuded mature pycnidiospores have been reported to lose their ability to germinate in about one month after they were produced (Kiely, 1948b). Darnell-Smith (1918) also showed that the rapidity with which spores germinate depended largely on the age of the spores (time since released from pycnidia) with young spores germinating within 12 h and older spores taking several days to germinate while many failed to germinate.

Abstract 
Declaration 
Acknowledgements 
Table of Contents 
List of Figures 
List of Tables 
Chapter 1 General introduction 
1.1 References
Chapter 2 Review of Guignardia citricarpa Kiely, the causal agent of citrus black spot 
2.1 The pathogen, Guignardia citricarpa
2.1.1 Origin and distribution of Guignardia citricarpa
2.1.2 Guignardia species on citrus
2.1.3 Morphology of Guignardia citricarpa
2.1.4 Sporulation
2.1.5 Spore germination
2.2 The host, Citrus
2.3 The disease, citrus black spot
2.3.1 Origin and distribution of citrus black spot
2.3.2 Economic importance of citrus black spot
2.3.3 Inoculum
2.3.3.1 Ascospores
2.3.3.2 Pycnidiospores
2.3.3.3 Symptomless infection
2.3.4 Infection
2.3.5 Symptoms
2.3.5.1 Fruit symptoms
2.3.5.1.1 Red spot
2.3.5.1.2 Hard spot
2.3.5.1.3 Freckle spot
2.3.5.1.4 Virulent spot
2.3.5.1.5 Speckled blotch
2.3.5.1.6 Cracked spot
2.3.5.2 Citrus tan spot
2.3.5.3 Leaf symptoms
2.3.5.4 Twig symptoms
2.3.6 Control
2.3.6.1 Chemical control
2.3.6.2 Non-chemical control
2.4 References
Chapter 3 Failure of Phyllosticta citricarpa pycnidiospores to infect Eureka lemon leaf litter 
3.1 Abstract
3.2 Introduction
3.3 Materials and methods
3.3.1 Pure culture
3.3.2 Infected fruit
3.3.3 Peelings of infected fruit
3.3.4 Polymerase chain reaction
3.3.5 Ascospore capturing
3.4 Results
3.5 Discussion
3.6 References
Chapter 4 Susceptibility of citrus leaves to Phyllosticta citricarpa relative to leaf age and phenolic acid content 
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.3.1 Leaf inoculation
4.3.2 Extraction and quantification of phenolic acids
4.3.3 Statistical analysis
4.4 Results
4.5 Discussion
4.6 References
Chapter 5 Monitoring Guignardia citricarpa ascospores from citrus leaf litter in commercial orchards
5.1 Abstract
5.2 Introduction
5.3 Materials and methods
5.4 Results
5.5 Discussion
5.6 References
Chapter 6 Artificial wilting of symptomless green leaves to enhance detection of Guignardia citricarpa 
6.1 Abstract 7
6.2 Introduction
6.3 Materials and methods
6.4 Results
6.5 Discussion
6.6 References
Chapter 7 Leaf litter management as a non-chemical means of reducing citrus black spot 
7.1 Abstract
7.2 Introduction
7.3 Materials and methods
7.4 Results
7.5 Discussion
7.6 References
Chapter 8 General discussion 
8.1 References
Appendix
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