Colletotrichum Species Associated with Citrus: Disease Biology, Global Diversity, Pathogenicity, and Biosecurity Implications

1. Introduction

The genus Colletotrichum represents important fungal plant pathogens globally and is also capable of causing disease in animals [1]. Currently, the classification system of Colletotrichum comprises 20 species complexes with more than 340 Colletotrichum species. While a few species can be host-specific, the majority infect a wide range of host species [1,2,3,4,5]. Until recently, the host range of Colletotrichum species encompassed over 760 plant species belonging to more than 30 plant genera in tropical, subtropical, and temperate regions [1,6,7]. Among the host species, dicotyledonous plants were the most frequently recorded, accounting for more than 77%, followed by monocotyledonous hosts (20%), gymnosperms (1.4%), and ferns and mosses, which were seldom recorded [1].

Colletotrichum species infect Citrus leaves, twigs, flower petals, and cause disease on immature crops and fruit before harvest, as well as on mature crop and fruit products post-harvest, negatively affecting yield quantity, fruit quality and marketability [2,8,9]. An epidemic of Colletotrichum in the field can easily lead to large losses in agricultural production and therefore cause significant economic losses for farmers [10]. Colletotrichum species are also isolated from healthy plant tissues and recognized as being in endophytic or latent life stages, or from dead plant tissues and recognized as saprobes [2,3]. Latent infection occurs when the pathogen enters the host tissue and remains inactive, then causes disease as the host’s physiology changes due to tissue maturation or exposure to abiotic stresses [2,3,11]. Latent infection is more frequently found in fruits, where the pathogen infects the fruit when it is immature and causes disease when the fruit starts to ripen, especially during the post-harvest period [12].

Citrus plants are perennial trees in the family Rutaceae that prefer to grow in areas with consistent sunlight and sufficient water [13]. Mandarin (C. reticulata), pomelo (C. maxima) and citron (C. medica) are the three ancestral species of the genus Citrus, and all commercially cultivated Citrus species are hybrids or introgressions between these three species [14,15]. As an important fruit crop worldwide, citrus is now mainly produced in Argentina, Australia, Brazil, China, Chile, Costa Rica, Egypt, the European Union, Guatemala, Israel, Japan, Mexico, Morocco, Peru, South Africa, Turkey, the United States, and Vietnam [16]. Grapefruit (Citrus × paradisi), lemon (Citrus limon), lime (Citrus aurantiifolia), tangerine/mandarin (Citrus reticulata) and orange (Citrus × sinensis) are the most popular species produced commercially [17]. Global citrus production for 2023/24 was estimated to be 102.6 million tons [16].

The high adaptability and wide host range of Colletotrichum species contribute to their persistence in citrus production regions. Accurate taxonomy of Colletotrichum species and knowledge of the pathogen’s pathogenic capacity on different hosts have important implications for plant biosecurity risk management and the development of integrated control strategies. Hence, this review aims to provide a critical assessment of the major Colletotrichum species associated with citrus diseases, their global diversity, distribution, and biosecurity implications, as well as to identify areas in need of further research.

2. Disease Cycle and Infection Strategies of Colletotrichum Species

Colletotrichum species have endophytic, latent or quiescent, hemibiotrophic, and necrotrophic lifestyles [2]. These fungi employ specialised infection strategies to infect host plants. Knowledge of the disease cycle and infection strategies of Colletotrichum species is essential for managing potential plant biosecurity risk and developing integrated control strategies.

Colletotrichum species favour warm temperatures between 25°C and 28°C with high air humidity, and become inactive under extremely low or high temperatures, low air humidity, or direct sunlight [2,18]. The pathogen survives unfavourable conditions, such as winter, by colonising plant seeds, twigs, leaf litter or alternative hosts. When environmental conditions become suitable, asexual fruiting structures (acervuli) are formed which release conidiospores however, in some species sexual fruiting structures (perithecia) also develop and release ascospores. Released spores spread to nearby plant organs through rain splash, wind, or irrigation, germinate, and form infection structures (appressoria). Penetration pegs are formed from the appressoria, where high mechanical pressure is applied to enable penetration of the host cuticle and epidermal cell walls [19,20,21,22]. Some species in the acutatum complex have also been reported to enter leaves through stomata or wounds [23,24].

After successful penetration, most Colletotrichum species infect host plants through a hemibiotrophic strategy, which begins with a biotrophic phase and then transitions to a necrotrophic phase [2,20]. During the biotrophic phase, infection vesicles and primary hyphae develop inside the infected cells without causing necrosis. Eventually, the pathogen transitions to the necrotrophic phase, during which secondary hyphae develop and spread to adjacent cells, leading to host tissue necrosis [25].

Colletotrichum species employ a diverse range of infection strategies to restrain host defence mechanisms and facilitate colonisation [26]. The cuticle is an important physical barrier of plants that prevents initial infection [27,28]. Appressorium formation is an important strategy for most Colletotrichum species. Glycerol and other osmolytes accumulate in the appressorium, generating substantial turgor pressure of up to 8 MPa, which forces a narrow penetration peg to mechanically penetrate the host cuticle. Mutant Colletotrichum species that form fewer appressoria have been shown to have decreased pathogenicity [29]. The production of cutinase is also a critical strategy during the early stages of infection. Auyong et al. (2015) found that Colletotrichum truncatum produced cutinase to assist the infection of chilli fruit and soybean leaves, and that the pathogenicity of the pathogen decreased when the cutinase gene was silenced [30]. After successful penetration, Colletotrichum species produce pathogen-encoded small secreted proteins (SSPs) to manipulate host metabolism or suppress defence signalling, thereby evading host immune responses during the initial biotrophic phase [31].

Another major defence mechanism of host plants is to enhance structural defence by reinforcing the cell wall, mainly through the accumulation of polysaccharides such as cellulose, hemicelluloses, and pectins [32]. In the citrus cell walls, lignin may accumulate after pathogen penetration to enhance resistance [27,33]. Gan et al. (2013) found that Colletotrichum gloeosporioides and C. orbiculare produce a large number of carbohydrate-active enzymes (CAZymes), including cellulose-degrading and pectin-degrading enzymes, to degrade these polysaccharides and assist hyphal expansion [34].

Some Colletotrichum species remain dormant after entering plant tissue and cause disease once the host is under stress or environmental conditions become suitable for the pathogen to complete its life cycle [11]. The physiological maturity of the host can play an important role in disease development, as physiological and biochemical changes reduce defence activity and increase sugar content, thereby facilitating infection by Colletotrichum species [35,36,37].

Quiescence is frequently observed in Colletotrichum infections of fruit, in which fungal development is temporarily arrested until the host tissue ripens or reaches maturity. Although active growth is limited during this phase, the pathogen produces fungal effectors to modify the host environment and maintain its survival, which is similar to an endophytic lifestyle. Colletotrichum gloeosporioides, C. acutatum, C. higginsianum, C. graminicola, and C. coccodes actively release ammonium to increase the environmental pH, alter plant gene expression, modulate host responses, and induce confined cell death, after which the pathogen transitions to the necrotrophic phase [37,38]. The transcription factor PacC regulates genes encoding transporters, antioxidants, and cell-wall degrading enzymes and is crucial for successful colonisation. Alkan et al. (2013) reported that PacC of C. gloeosporioides activates degradative enzymes under alkaline conditions [39]. O'Connell et al. (2012) found that C. higginsianum and C. graminicola contain more genes encoding key enzymes involved in secondary metabolism than Pyricularia (Magnaporthe) oryzae and Fusarium graminearum. Some of these genes are essential for maintaining primary and secondary metabolism and encode mycotoxins that are important for pathogenicity [40].

The nature of infection by Colletotrichum species through the formation of appressoria and penetration of the cuticle and epidermal cells is important in the design of bioassays to determine pathogenicity. As discussed later a bioassay to assess the ability of Colletotrichum isolates to cause infection must not be based solely on wounding the cuticle and epidermis before placing the inoculum over the wound site.

3. Citrus Diseases Associated with Colletotrichum Species

Globally, citrus diseases caused by Colletotrichum species have been reported from every continent. Anthracnose, twig and shoot dieback or withertip, post-bloom fruit drop (PFD), and Key lime anthracnose are important citrus diseases associated with Colletotrichum species [41,42,43,44,45].

3.1. Anthracnose

Anthracnose is the most typical citrus disease associated with Colletotrichum species and affects citrus both pre- and post-harvest. To date, anthracnose has been reported globally from Citrus × aurantium, Citrus × latifolia, Citrus × sinensis, Citrus aurantiifolia, Citrus aurantium, Citrus australasica, Citrus bergamia, Citrus digitata, Citrus flamea, Citrus floridana, Citrus grandis, Citrus hystrix, Citrus japonica, Citrus latifolia, Citrus limon, Citrus maxima, Citrus medica, Citrus nobilis, Citrus paradisi, Citrus reticulata, Citrus nobilis × Citrus deliciosa, and the mandarin-like hybrid ‘Mandared’ (Table 1).

As a pre-harvest disease of citrus, anthracnose infects leaves, stems, flower petals, and unharvested fruit, with notable symptoms including sunken necrotic lesions and concentric rings of conidial masses [46,47]. Symptoms on leaves include necrotic lesions, greasy spots, mesophyll collapse, or blight, leading to wilting, yellowing, and leaf drop [42,48,49,50,51,52,53]. Necrotic spots, canker, or splitting are common symptoms on stems, branches, and petioles [45,49]. Fruits infected by anthracnose often develop sunken, brown or black tear stains, spots, or lesions on the rind. Yellowing of immature fruit, rind russeting and microcracking, and fruit dryness have also been reported as anthracnose symptoms on citrus fruit [42,48,49,52,53,54,55,56,57]. Stem end rot, in which the disease progresses downwards from infected twigs to the stem end of the fruit, is a severe pre-harvest citrus anthracnose disease. It causes circular, brown, soft to semi-pliable lesions at the stem end of the fruit, leading to fruit rot, fruit drop, and fruit mummification. Infection has also been detected at both the stem and stylar ends of the fruit [44,45].

Post-harvest anthracnose refers to anthracnose that develops during post-harvest storage, transport, or marketing. Colletotrichum species remain quiescent after entering immature citrus fruit and cause anthracnose when the fruit starts to ripen, especially during the post-harvest period [2,12]. The fruit becomes more suitable for disease development during physiological maturation and post-harvest storage due to a favourable environmental pH range of 5.8 to 6.5, softening of the rind, and increased soluble carbohydrate content. Sunken, black spots and tear stains are common early-stage symptoms, and the lesions continue to expand and eventually become firm and dry as the fruit becomes overmature or is exposed to stress. Severely infected fruit becomes soft, and orange conidial masses appear at the centre of the lesions [36].

The economic impact caused by anthracnose is profound. Citrus fruit losses can be as high as 90-100% when environmental conditions are suitable for infection [58]. Severe pre-harvest anthracnose symptoms on orange fruit were observed from 2010 to 2013 in Italy [59]. Cheng et al. (2013) reported that leaf drop and fruit spots caused an average yield loss of 15% in C. reticulata cv. Shiyue Ju in China in 2008 [60]. Fruit drop caused by stem end rot led to significant economic losses in Kinnow mandarin production by negatively affecting fruit yield and quality [44]. For post-harvest anthracnose, an estimated infection incidence of 15% was observed in grapefruit in Mexico during June 2019 [61]; approximately 10% of orange fruit were infected in China in July 2017 and 2018 [62]; and about 15% of Key lime fruit were infected in China in 2016 [63].

3.2. Twig and Shoot Dieback or Withertip

Twig and shoot dieback, which is also known as citrus withertip, has attracted increasing attention in recent years [51,64]. Twig dieback has been frequently reported from multiple commercial citrus species and varieties, including Citrus × aurantium, Citrus × latifolia, Citrus margarita (syn. Fortunella margarita), Citrus × sinensis, Citrus × tangelo, Citrus trifoliata (syn. Poncirus trifoliata), Citrus aurantiifolia, Citrus limon, Citrus maxima, Citrus medica, Citrus paradisi, Citrus reticulata and the mandarin-like hybrid ‘Mandared’ (Table 1).

Typical symptoms of twig dieback include gumming on twigs, twig blight, twig and branch dieback, crown thinning, leaf chlorosis, leaf necrosis, and defoliation [42,46,51,64,65]. The disease starts from the tips of the twigs, spreads towards the main stem, and eventually infects the whole plant, negatively affecting normal plant growth, structure, and function, and potentially leading to plant death [46,66,67]. An outbreak of severe twig dieback resulted in a 40% yield loss in Citrus reticulata cv. Kinnow in Pakistan in 2014 [65]. Moreover, twig dieback caused by Colletotrichum spp. is likely to be a source of inoculum that spreads to flower petals and then to young fruit, leading to post-harvest anthracnose [46,53]. The pathogen can survive in leaves and twigs for a long period before flowers are present [68]. Camiletti et al. (2022) reported latent infection of Colletotrichum dieback on citrus leaves and twigs in California [64].

3.3. Post-Bloom Fruit Drop

Post-bloom fruit drop (PFD), or blossom blight, is another important citrus disease caused by Colletotrichum species. It was initially reported in Belize and is now widely distributed in tropical and subtropical regions of the Americas [69,70,71,72,73,74,75]. Colletotrichum species cause PFD in all commercial citrus varieties, while sweet orange is the most common host species [69,73,75,76].

Post-bloom fruit drop develops quickly under highly humid conditions during the bloom period, and disease symptoms appear within 48 to 72 hours after initial infection [75]. Peach-to-brown, water soaked, elongated lesions first appear on the petals and filaments of diseased flowers. The lesions continuously expand, causing blight of the whole flower and eventually infecting the entire flower cluster. As the disease develops, the pathogen enters the fruit, resulting in yellowing and abscission of the developing fruit [74,75]. Persistent calyces remaining attached to the peduncle are an important and specific symptom for diagnosing PFD [42,75,76]. The observation of petal lesions alone on citrus plants is considered pre-harvest anthracnose [48,52].

Severe outbreaks of PFD cause extensive losses of immature citrus fruit. Fruit losses of sweet orange, grapefruit, lemon and Tahiti lime ranged from 25–35% in Bermuda [75]. In Brazil, PFD may cause severe yield losses of up to 93% when the bloom period coincides with rainy conditions [76].

3.4. Key Lime Anthracnose

Key lime anthracnose is a specific disease complex affecting only the leaves, twigs, flowers and fruit of Key lime [42,70,77]. Common symptoms include necrotic lesions on leaves, twigs, flowers, and fruit, ranging from small lesions to necrotic blight of entire shoots and inflorescences [71,78]. The disease has been observed in citrus orchards in Belize, Brazil, Costa Rica, Mexico, Panama, the Dominican Republic, and the USA, and has been suggested to be limited to the Americas [48,71,78].

4. Global Diversity and Distribution of Citrus-Associated Colletotrichum Species

Thirty-nine Colletotrichum species have been isolated from a range of Citrus species globally. Most Colletotrichum species have been reported as plant pathogens, while several species have been found to exhibit endophytic or latent lifestyles, or have been observed as saprobes (Table 1). In Colletotrichum, a monophyletic clade formed by a group of species exhibiting similar morphological characteristics is defined as a species complex, or aggregate [5,79].

4.1. Gloeosporioides Complex

The gloeosporioides complex is the most commonly reported species complex associated with citrus. Sixteen species belonging to the gloeosporioides complex have been reported as pathogens, endophytes, or saprobes on citrus (Table 1).

Colletotrichum gloeosporioides is the most common Colletotrichum species associated with citrus globally [42,43,46]. The species was originally isolated from orange (Citrus × sinensis) in southern Italy and has been found to be the most prevalent pathogen causing pre- and post-harvest anthracnose of multiple Citrus species and varieties in Australia, China, and Europe [5,42,49,54]. Previously, C. gloeosporioides was reported to cause leaf anthracnose in Australia, China, Greece, Indonesia, Italy, Mexico, Portugal, Spain, Thailand, Tunisia, and Turkey [42,45,48,49,50,51,52,53,57,59,80,81,82]; flower anthracnose in Portugal, Spain, and Tunisia [42,48,52]; branch anthracnose in Portugal [48]; stem spot in Australia [49]; and pre- and post-harvest fruit anthracnose in Australia, Brazil, China, Ghana, Greece, Indonesia, Italy, Mexico, Morocco, Portugal, South Africa, Thailand, Tunisia, Turkey, Vietnam, and Zimbabwe [42,45,48,49,52,53,54,55,56,57,59,61,63,80,81,82,83]. Colletotrichum gloeosporioides has also been reported as the most prevalent species causing citrus twig dieback in Australia and Italy and has been associated with citrus twig dieback or withertip in Algeria, China, Greece, Malta, Morocco, Pakistan, Portugal, Spain, Thailand, Tunisia, Turkey, and the USA [42,45,46,51,52,53,54,56,58,67,80,81,84,85,86]. In Brazil, Bermuda, and Mexico, C. gloeosporioides was found to cause post-bloom fruit drop of Citrus limon and Citrus × sinensis [57,69,73,75,76]. Moreover, C. gloeosporioides has also been found to exhibit an endophytic lifestyle in asymptomatic branches and leaves in China and latent infection in asymptomatic tissues in the USA [43,64,87]. Saprobic C. gloeosporioides has been found on dead fallen leaves in Australia, Malta and China [42,43,49].

Colletotrichum siamense is the second most important species in the gloeosporioides complex. The species has been reported to cause citrus fruit anthracnose in Australia, Bangladesh, China, Egypt, India, Thailand, and Vietnam [44,45,49,54,60,80,88]; leaf anthracnose in China and Thailand [45,60,80,85]; branch canker in Thailand [45]; and twig dieback or withertip in Pakistan and Thailand [45,65,67,80]. Colletotrichum siamense has also been reported as an endophyte isolated from asymptomatic leaves in China [87,89].

Colletotrichum fructicola is the third most common species in the gloeosporioides complex. Globally, C. fructicola has been found to infect citrus leaves in China and Thailand [45,50,80], branches in Australia [49], twigs in Tunisia [53], and fruit in Australia and China [49,62]. Endophytic C. fructicola has also been isolated from asymptomatic leaves in China [43,87].

In addition, C. australianum was associated with fruit anthracnose in Australia [49]. Colletotrichum endophyticum was found to cause leaf spot in Thailand, as well as fruit and leaf anthracnose in Indonesia [45,82]. Colletotrichum helleniense was found to infect twigs and fruit in Greece [42]. Colletotrichum hystricis and C. kahawae subsp. ciggaro (C. cigarro) were reported to cause leaf disease in Italy [42,90]. Colletotrichum queenslandicum was associated with fruit anthracnose in Indonesia and leaf anthracnose in Indonesia and the USA [82,91]. Colletotrichum syzygicola was reported to cause fruit anthracnose in Thailand [92]. Colletotrichum theobromicola was associated with fruit anthracnose in Australia [49]. Colletotrichum aenigma was isolated from Citrus × sinensis in Italy [93]. An unidentified Colletotrichum species was found to infect citrus leaves and fruit in Australia [49]. Colletotrichum tainanense and C. tomentosae were found as endophytes in asymptomatic leaves in China, while C. asianum was found as an endophyte in asymptomatic twigs in China [89].

4.2. Boninense Complex

The boninense complex is the second most commonly reported species complex associated with citrus globally. Seven species belonging to the boninense complex have been reported as pathogens, endophytes, or saprobes on citrus (Table 1).

Colletotrichum karstii is the most important species in the boninense complex and the second most prevalent Colletotrichum pathogen associated with citrus globally. Colletotrichum karstii has frequently been found to cause pre- and post-harvest anthracnose on multiple Citrus species and varieties across a wide geographical distribution, including leaf anthracnose in Australia, China, Italy, Portugal, Spain, and Turkey [42,43,48,49,50,59,81]; branch anthracnose in Portugal [48]; and pre- and post-harvest fruit anthracnose in Italy, Portugal, South Africa, Tunisia, and Turkey [42,48,52,54,59,81,94]. Colletotrichum karstii has also been associated with citrus twig dieback or withertip in Australia, Italy, Malta, Portugal, Spain, Turkey, and the USA [42,51,81,85,86,94]. Moreover, C. karstii has been reported to exhibit an endophytic lifestyle in asymptomatic branches, leaves and shoots in China, as well as latent infection in asymptomatic tissues in the USA [43,64,87].

Colletotrichum novae zelandiae is the second most important species in the boninense complex. The species has been reported to cause citrus twig dieback or withertip in Australia and Malta [42,86], leaf lesions in Greece and Malta [42], and fruit infection in New Zealand [95].

Colletotrichum catinaense is the third most common species in the boninense complex and has been found to cause twig withertip and leaf lesions in Italy, as well as fruit tear stain in Portugal [42].

In addition, C. boninense has been reported to cause leaf anthracnose in China [50], C. constrictum has been associated with fruit rot in New Zealand [95], and C. limonicola has been found to cause leaf lesions in Malta [42]. Colletotrichum citricola has been reported to exhibit an endophytic lifestyle in asymptomatic leaves and have been reported as saprobes on leaves in China [43,87].

4.3. Acutatum Complex

The acutatum complex is the third most commonly reported species complex associated with citrus. Seven species belonging to the acutatum complex have been reported as pathogens on citrus (Table 1).

Colletotrichum abscissum has been reported as the causal agent of post-bloom fruit drop in Brazil and the USA [69,72,73,74,75,76]. This species was previously identified as C. acutatum and was therefore recorded as being associated with post-bloom fruit drop under that name [75,76]. It was later reclassified as C. abscissum [72].

Colletotrichum limetticola has been found to cause Key lime anthracnose in the USA [70]. This species was initially identified as C. acutatum and was therefore recorded as being associated with Key lime anthracnose under that name [75,78]. It was later reclassified as C. limetticola [70].

In addition, C. johnstonii has been associated with citrus fruit rot in Australia and New Zealand [70,96], C. acutatum has been reported to cause leaf lesions in Italy [42], C. godetiae has been found to infect the fruit of Citrus aurantium [70], C. citri has been reported to cause shoot anthracnose in China [43], and C. simmondsii has been associated with fruit anthracnose in China [9].

4.4. Magnum Complex

Three species belonging to the magnum complex have been reported as pathogens or endophytes on citrus (Table 1). Colletotrichum brevisporum has been found to cause leaf anthracnose and to exhibit an endophytic lifestyle in China [5,50]. Colletotrichum guangdongense has been found as an endophyte in asymptomatic twigs in China [89]. Colletotrichum kokhaense has been associated with leaf lesions in Thailand [80]

4.5. Gigasporum Complex

Two species belonging to the gigasporum complex have been reported as pathogens or endophytes on citrus (Table 1). Colletotrichum gigasporum has been reported to cause twig dieback and leaf lesions in Thailand [80]. The species has also been found as an endophyte in asymptomatic leaves in China [87]. Colletotrichum citri-maximae has been found to exhibit an endophytic lifestyle in asymptomatic fruit in China [54].

4.6. Dracaenophilum, Orchidearum and Truncatum Complexes

Colletotrichum tropicicola, belonging to the dracaenophilum complex, has been reported to cause leaf lesions and twig dieback in Thailand [80,97]. Endophytic C. tropicicola has also been isolated from asymptomatic leaves in China [87]. Colletotrichum tropicicola has also been isolated from Citrus sp. in Mexico [98].

Colletotrichum plurivorum, belonging to the orchidearum complex, has been reported to infect citrus leaves in Thailand [45,80]. The species has also been found as an endophyte in asymptomatic leaves in China [87,89]. Colletotrichum truncatum, belonging to the truncatum complex, has been found to cause shoot and leaf anthracnose and to exhibit an endophytic lifestyle in asymptomatic leaves in China [43,87].

4.7. Singleton Species

The singleton species C. citrus-medicae has been reported to cause leaf spot of Citrus medica in China [99].

These identified citrus-associated Colletotrichum species have important implications for plant biosecurity risk management and the development of integrated control strategies. However, some of these Colletotrichum species may have been misidentified by analysing only morphological characters [56], only ITS sequences [52,53,59,84], or species-specific primers designed before the adoption of multi-locus phylogeny [75,76]. Phylogenetic analyses based on the combination of ITS and gapdh sequences [55] or ITS and tub2 sequences [93] are also unable to clearly distinguish a Colletotrichum species from closely related species [5]. Further identification of Colletotrichum species should utilise the polyphasic approach, which consists of multi-gene phylogenetic analysis, traditional morphological character descriptions, and other information such as geographical and ecological data.

5. Pathogenicity and Host- or Tissue-Specificity of Citrus-Associated Colletotrichum Species

Pathogenicity, which indicates the ability of a pathogen to cause disease on a host, consists of variations in aggressiveness and pathotype differences. Different Colletotrichum species may contain isolates with various levels of aggressiveness and different pathotypes on the same host species [42,45]. Different pathotypes within the same Colletotrichum species can be identified on the same host species according to qualitative differences in disease expression [100,101].

Controlled environment bioassays using detached or in planta leaves, twigs, flower petals, and fruit are widely used to test the pathogenicity of Colletotrichum species. Many pathogenicity tests have been performed on Citrus by inoculating Colletotrichum isolates onto the same tissues of the same host species to reproduce similar disease symptoms and then re-isolating the pathogens from the inoculated tissues [88,96]. Studies have reported that Colletotrichum isolates obtained from one host or tissue may also cause symptoms on other hosts or tissues, suggesting that these species are not strictly host- or tissue-specific. However, these findings are often dependent on the type of bioassay. Cross-host inoculation bioassays have been conducted to test the pathogenicity of Colletotrichum species associated with citrus disease on multiple hosts and tissues, and several species have demonstrated the ability to infect citrus hosts or tissues beyond the original host tissue using wound or non-wound inoculation (Table 2).

Similarly, Colletotrichum species have been shown to infect and cause disease across plant genera. Colletotrichum acutatum isolated from diseased fruit of Olea europaea and C. karstii isolated from diseased leaves of Camellia sp. caused disease symptoms on wounded in planta twigs of sweet orange ‘Tarocco Scirè’, lemon ‘Femminello 2Kr’, and bergamot ‘Fantastico’; wounded detached leaves of sweet orange ‘Moro’ and ‘Navelina’; and wounded fruit of apple (Malus domestica) ‘Fuji’ and ‘Cripps Pink’. The C. acutatum and C. karstii isolates caused significantly larger lesion areas on wounded leaves of sweet orange ‘Navelina’ than those caused by Colletotrichum isolates associated with citrus disease [51]. Colletotrichum gloeosporioides isolated from diseased mandarin and sweet orange twigs, diseased sweet orange leaves, and diseased lemon fruit, as well as C. karstii isolated from diseased sweet orange twigs, infected wounded fruit of apple ‘Fuji’ and ‘Cripps Pink’ [51]. Colletotrichum gloeosporioides associated with lemon twig withertip caused lesions on wounded fruit of apple ‘Gala’, avocado ‘Fuerte’, pear ‘Santa Maria’, pepper ‘Capia’, and banana ‘Cavendish’ [81]. Colletotrichum karstii causing kumquat twig and branch dieback infected wounded fruit of apple ‘Golden Delicious’ [102]. These results may however overstate the seriousness of cross-host pathogenicity because the bioassays involved wounding the cuticle and epidermis of the plant tissue with a sterile needle or scalpel before placing the inoculum over the wound.

The cuticle and epidermis are important physical barriers that defend the host against pathogen infection. Artificial damage to these physical barriers bypasses the need for Colletotrichum species to penetrate the cuticle and epidermis and may therefore provide a biased measure of pathogenicity [27,28,100,103]. In contrast, during non-wound inoculation, the inoculum is placed directly on the surface of the host tissue, requiring the Colletotrichum species to penetrate the physical barriers to initiate infection. This method is closer to infection occurring in the natural environment [2,101,103]. Hence, the non-wound inoculation method tends to provide a more realistic evaluation of the pathogenicity of weakly aggressive species or species with latent or endophytic lifestyles [100,101]. Colletotrichum gigasporum, C. kokhaense, C. gloeosporioides, C. plurivorum, and C. siamense showed various degrees of aggressiveness on non-wounded lime and orange fruit but showed similarly high levels of aggressiveness when the citrus fruit was wounded before inoculation [80]. Colletotrichum gloeosporioides, C. karstii, and C. novae-zelandiae, which developed a latent lifestyle after infecting non-wounded orange and lemon twigs, became pathogenic when the citrus twigs were inoculated using the wound inoculation method [86].

6. Biosecurity Implications of Citrus-Associated Colletotrichum Species

Thirty-nine Colletotrichum species have been found to be associated with Citrus species, with additional novel species continuing to be described [1,5]. Most Colletotrichum species have been collected from a range of Citrus species and host tissues, except for species represented by only one isolate. Host- or tissue-specific pathogens typically have a more restricted distribution, whereas those with a broad host range are more likely to cause disease worldwide. Colletotrichum gloeosporioides, which is the most important Colletotrichum species associated with citrus globally, has also been reported from the greatest diversity of Citrus species and can infect all citrus tissues [42,43,46,73]. Isolates of many citrus-associated Colletotrichum species, such as C. fructicola, C. gigasporum, C. gloeosporioides, C. karstii, C. plurivorum, C. siamense, C. theobromicola, and C. tropicicola, have been reported from hosts belonging to different genera [1,104,105,106,107]. Cross-plant genus infection by Colletotrichum species has also been demonstrated on wounded fruits [9]. Both in planta and detached tissue bioassays demonstrated that C. acutatum and C. karstii isolates obtained from hosts belonging to different genera were able to infect citrus using wound inoculation [51]. The isolation of the same Colletotrichum species from different hosts, together with the first reports of Colletotrichum species on citrus in different geographical locations, demonstrates the potential for cross infection and poses a threat to the biosecurity of the citrus industry.

In addition, all species in the genus Citrus are hybrids or introgressions between mandarin (Citrus reticulata), pomelo (Citrus maxima) and citron (Citrus medica) [14,15]. The genetic similarity among commercially cultivated Citrus species may be conducive to the transmission of Colletotrichum species between different citrus hosts. Meanwhile, the demonstrated cross-host infection by C. gloeosporioides isolated from citrus on wounded fruit of apple, avocado, pear, pepper, and banana, and by C. karstii isolated from citrus on wounded apple fruit, makes these two species potential high-risk pathogens for different fruit and vegetable industries, especially where susceptible plants are grown near citrus [51,81,102].

An exotic pathogen may rapidly colonise a new environment and pose a biosecurity risk to local plants when environmental conditions are suitable for the species [108]. The temperature tolerance of Colletotrichum species is an important factor when considering their distribution and the damage they may cause to the local ecosystem. Although the optimum temperature for mycelial growth, conidial germination, and appressoria formation and melanisation of C. gloeosporioides was estimated to be around 24°C, its growth rate remained relatively high at 30°C and was only moderately reduced at 35°C compared with its growth at 25°C [51,64,76]. The broad temperature tolerance of C. gloeosporioides is consistent with its reported occurrence across a wide range of climates, including tropical, subtropical, Mediterranean, temperate, and semi-arid regions (Table 1).

Colletotrichum siamense was originally described from Arabica coffee (Coffea arabica) in Thailand and has been reported to grow optimally at 30°C. Consistent with this relatively high optimal growth temperature, previous reports of C. siamense from diseased citrus tissues have mainly occurred in tropical climate zones, and C. siamense has been identified as the most prevalent Colletotrichum species associated with citrus in Thailand [1,80,109]. Furthermore, C. acutatum and C. karstii have optimum growth temperatures of around 24°C, and their growth rates were dramatically reduced at 30 and 35°C, suggesting that these pathogens may be more likely to cause disease on plants in temperate regions [51,64,76]. In contrast, C. endophyticum, C. fructicola, C. gigasporum, C. plurivorum, and C. tropicicola may cause severe damage to agricultural industries in tropical climate zones [43,45,49,50,80,82,89,97]. Therefore, Colletotrichum species may need to be listed as quarantine pathogens by national biosecurity regulatory authorities in countries or states where they have not previously been detected, to reduce potential biosecurity risks to local plants, especially in areas with environmental conditions suitable for these species.

Colletotrichum species have multiple lifestyles and can transition to different lifestyle stages during the disease cycle to complete infection [2,11]. Several Colletotrichum species associated with citrus diseases, including C. fructicola, C. gloeosporioides, C. siamense, C. karstii, C. brevisporum, C. gigasporum, C. tropicicola, C. plurivorum, and C. truncatum, have been reported as either causing citrus disease or exhibiting endophytic or quiescent lifestyles (Table 1). Although several Colletotrichum species have only been observed as endophytes in citrus, their pathogenic potential cannot be excluded. Colletotrichum gigasporum and C. plurivorum were first identified as endophytes and were later reported to cause citrus disease [45,80,87,89]. Camiletti et al. (2022) demonstrated that C. gloeosporioides and C. karstii isolated from asymptomatic leaves and twigs were able to cause twig dieback [65] . Therefore, the potential dispersal of Colletotrichum species in asymptomatic citrus tissues is of major importance when considering biosecurity. Endophytic or quiescent Colletotrichum species may remain undetected and be overlooked during quarantine inspection when citrus fruit or plant material is imported. In addition, C. citricola and C. gloeosporioides have been observed as saprobes on dead fallen leaves, which may serve as potential sources of inoculum for citrus and other hosts and pose biosecurity risks both locally and during transportation to other regions [42,43,49].

Rapid and accurate identification of Colletotrichum species is particularly important in quarantine inspection [10]. Real-time quantitative PCR, which enables the rapid, sensitive, and accurate quantification of target fungal DNA in a wide range of environmental samples, has been developed for several Colletotrichum species. De Silva (2019) developed a qPCR assay to differentiate C. scovillei from 14 closely related Colletotrichum species, especially C. acutatum and C. nymphaeae that cause anthracnose in chili [110]. Debode et al. (2009) quantitatively detected C. acutatum in naturally infected and symptomless strawberry leaves using real-time PCR [111]. A real-time PCR assay has also been developed for the rapid and accurate identification of C. coccodes in symptomatic tomato leaves and external asymptomatic potato tubers [112]. Azevedo-Nogueira et al. (2021) designed a species-specific qPCR assay to enable the rapid detection and quantification of C. acutatum sensu stricto in pulverised asymptomatic olive fruit at 16 hours post inoculation [113]. Additionally, knowledge of the resistance levels of multiple host varieties to the same pathogen species is essential for breeding resistant varieties for integrated disease management and minimizing both economic losses and environmental impacts [24]. Real-time quantitative PCR could also be used to screen resistant host varieties [114,115]. Therefore, real-time quantitative PCR assays could be developed for highly aggressive citrus-associated Colletotrichum species and applied in screening procedures for imported produce and in breeding programs to strengthen plant biosecurity. The problem with developing a PCR diagnostic technique is selecting species-specific primers due to the ever-increasing number of new species especially closely related pathogenic species.

Globally, records of new Colletotrichum species associated with citrus continue to increase, placing greater pressure on plant biosecurity risk management. Accurate identification of species is the initial step, as many species have been isolated solely from one host species in one geographic location. Further studies are urgently needed to provide a more comprehensive understanding of the host ranges and suitable environmental conditions of Colletotrichum species. Colletotrichum species have shown various levels of aggressiveness and pathotype differences when infecting the same host plant tissue under the same environmental conditions [42,43,45,48,52,53,80,81,86,101]. The pathogenicity of each Colletotrichum species indicates its potential to reduce productivity in citrus orchards. Detached organ assays are useful for rapid screening, require less space than in planta assays conducted in greenhouses or fields, and can produce disease symptoms similar to those observed following in planta inoculations [48,116]. Therefore, comprehensive pathogenicity bioassays that include common hosts of Colletotrichum species and a range of temperature and humidity conditions can be used to determine host range, estimate the potential distribution of newly reported species, and assess their potential to pose biosecurity risks to citrus trees, native plants, and crops.