**2. Endophytic Occurrence of Citrus Pathogens**

The agent of citrus black spot (CBS) *Phyllosticta citricarpa*, also known under the teleomorph name *Guignardia citricarpa* (Dothideomycetes, Botryosphaeriaceae), is one of the most noxious pests of these crops in subtropical regions, and it is subject to phytosanitary restrictions by the European Union and the United States. The employment of biomolecular methods has provided substantial support to the distinction between pathogenic isolates, typically slow-growing in axenic cultures and producing a yellow halo on oatmeal agar, and non-pathogenic isolates, which are morphologically similar but fast-growing, and producing conidia embedded within a thicker mucoid sheath [4–8]. The latter, characterized as a different species (*Phyllosticta capitalensis*), are known to be ubiquitous as endophytes in woody plants, having been reported from at least 70 botanical families [6,9,10]. *Guignardia endophyllicola*, treated as a separate species in a work also emphasizing its widespread endophytic occurrence [11], is at present recognized as a synonym. Differences between the two sister species also concern their metagenetic cycle. In fact, it has been ascertained that *P. citricarpa* is heterothallic, while *P. capitalensis* is homothallic [8]. This consolidated taxonomic distinction supports the exclusion from quarantine measures of plant material harbouring *P. capitalensis*. To this purpose, several rapid PCR assays have been developed [12–20]. The applicative use of these assays has enabled to exclude the presence of the pathogen in New Zealand, unlike what was previously assumed [21], and has supported the hypothesis of the possible endophytic occurrence of *P. citricarpa* in asymptomatic *Citrus* spp., as pointed out by several investigations (Table 1). Moreover, the two species have been clearly differentiated on account of their enzymatic profiles, with a higher expression of amylases, endoglucanases, and pectinases in *P. citricarpa*, suggesting a likely involvement of these enzymes in the pathogenic aptitude of the CBS agent [22]. Differences in terms of pathogenesis-related proteins have been confirmed after the genome sequencing of the two species, disclosing a higher number of coding sequences in *P. citricarpa* (15,206 versus 14,797). Such a difference has been interpreted considering the presence of growth and developmental genes involved in the expression of pathogenicity [23].

The issue of detection of contaminated material imported from areas where the pathogen is endemic has also prompted investigations concerning the assortment of *Phyllosticta* spp. able to colonize citrus plants in either symptomatic or latent courses. Several revisions have been published [17,24], and novel species characterized, which consistently enlarge the citrus-associated consortium within this widespread genus. 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. and growing conditions, has disclosed *P. paracapitalensis* to be even more widespread than *P. capitalensis*. Strains of both species were confirmed to be non-pathogenic on fruits under field conditions, and displayed antagonistic effects against the CBS agent, introducing their possible exploitation in the integrated management of this disease [31]. In this respect, it has been speculated that, rather than depending on intrinsic genetic factors, resistance to CBS by *C. latifolia* could be due to its systematic colonization by *P. capitalensis*, as disclosed by a dedicated investigation carried out in Brazil [32].

*Colletotrichum* (Sordariomycetes, Glomerellaceae) is another important ascomycete genus in course of coherent taxonomic revision. Besides *Colletotrichum gloeosporioides*, the agent of citrus anthracnose, it includes many species known for their endophytic aptitude. A recent investigation carried out in China on several *Citrus* spp. has shown a high proportion of endophytic strains to belong to *C. gloeosporioides sensu stricto*, calling for further investigations concerning the asymptomatic occurrence of this pathogen in citrus orchards. Additional identified species are *Colletotrichum fructicola* from Citrus reticulata cv. Nanfengmiju and *Citrus japonica* (=*Fortunella margarita*), and *Colletotrichum karstii* [33]. A similar widespread occurrence of C. gloeosporioides has been more recently confirmed in Brazil, where just one out of 188 isolates was found to be able to induce post-bloom fruit drop. This syndrome is more frequently associated to the species *Colletotrichum abscissum*, which, however, does not display an endophytic habit [34]. Endophytic C. gloeosporioides were also previously reported from Citrus limon in Argentina [35] and Cameroon [36].

One more meaningful example of endophytic fungus converting to pathogenic when plants are exposed to stress factors is represented by another member of the Botryosphaeriaceae, Lasiodiplodia theobromae. Characterized by a widespread endophytic occurrence [37,38], this species has been reported to exacerbate pre-harvest fruit drop and post-harvest fruit decay in plants of Citrus sinensis hit by the huanglongbing syndrome [39].

A quite intricate case deserving further investigations with reference to the epidemiological impact by endophytic strains is represented by members of the genus Diaporthe (Sordariomycetes, Diaporthaceae), also known under the anamorph name Phomopsis [40,41], which are widespread in different ecological contexts [41,42]. Besides the longtime known D. citri, more species in this genus have been recently identified as the causal agents of melanose, stem-end rot, and gummosis on *Citrus* spp., particularly, D. citriasiana and *D. citrichinensis* in China [43], and *D. limonicola*, *D. melitensis*, *D. baccae*, *D. foeniculina*, and *D. novem* in Europe [44]. Even more species have been reported for their endophytic occurrence as a result of a phylogenetic reassessment carried out in China, with eight known (D. arecae species complex, *D. citri*, *D. citriasiana*, *D. citrichinensis*, *D. endophytica*, *D. eres*, *D. hongkongensis*, and *D. sojae*) and seven new species (*D. biconispora*, *D. biguttulata*, *D. discoidispora*, *D. multiguttulata*, *D. ovalispora*, *D. subclavata*, and *D. unshiuensis*) [45].


**Table 1.** Endophytic fungi reported from *Citrus* species.

**Table 1.** *Cont.*


**Table 1.** *Cont.*


<sup>1</sup> Species are reported according to the latest accepted name, which might not correspond to the one used in the corresponding reference. <sup>2</sup> Conforming to the principle 'one fungus—one name' [66], the older genus names *Diaporthe* and *Phyllosticta* have been considered to deserve priority over *Phomopsis* and *Guignardia*, respectively. \* Novel species described for the first time from this plant source.

Endophytic occurrence has also been reported for other citrus pathogens, such as the leaf-spot agents *Alternaria alternata* [35,46–48] and *Alternaria citri* [49], *Fusarium oxysporum* [48], and *Fusarium sarcochroum*, known as a possible agent of dieback of twigs on mandarin and lemon [50]. The latter study also introduces new *Fusarium* spp. (*F. citricola, F. salinense, F. siculi*), causing cankers on several citrus species. Considering that pathogenic *Fusaria* often present an early latent stage, this finding claims for further assessments concerning their possible endophytic occurrence. Finally, it is worth mentioning *Physoderma citri*, a species ascribed to the *phylum* Blastocladiomycota reported to cause vessel occlusion, but also found in asymptomatic plants of several *Citrus* spp. [51].
