*2.1.1 Alternariosis*

*Alternaria* genus is the etiological agent of black spot and black heart diseases, even though the latter is more hazardous to human health. Indeed, *Alternaria* spp. produce more than 30 mutagenic or genotoxic mycotoxins, among which the most relevant are alternariol, alternariol monomethyl ether, altenuene, altertoxins, tentoxin, and tenuazonic acid [24]. Distinguishable symptoms feature both diseases. Black spot symptoms are necrotic areas in pomegranate rind, which appear as small circular black blotches, reddish-black in the middle, and surrounded by yellowishgreen halos. Internally fruit are healthy and edible (**Figure 1A**). On the other hand, pomegranates affected by black heart are apparently asymptomatic, but the inner part is rotted. Often, black-heart alternariosis is related to darker rind color and/ or lighter weight due to dehydration and aril disintegration; occasionally fruit may appear asymmetric and irregularly shaped. Internally, brown and soft-rotted arils become grayish-black in color (**Figure 1B**,**C**). Infection symptoms spread from the calyx area (crown) to the entire fruit along the carpel via; this diffusion pathway is shared by all latent fungal pathogens. Alternariosis is one of the most widespread pomegranate diseases, and these have been reported all over the world: Italy, Israel,

#### **Figure 1.**

*External symptoms of (A) black spot and (B) black heart caused by* Alternaria *spp. (C) Close-up on heart rot showing mycelium growth. Colonies on PDA of* Alternaria alternata*: (D) dark and (E) whitish strains. (F) Colony of* Alternaria arborescens *on PDA [6].*

Greece, California, Spain, and India. Species involved in Alternaria diseases belong to *A. alternata* and *A. arborescens* species-complex, their distribution is about overlapped; in addition, just in India is recorded *A. burnsii* [13, 25–29]. Although, within this genus conidial morphology is easily recognizable, the sporulation pattern is different among species and/or morphotypes, but intraspecific variability of colony morphology is high (**Figure 1C**-**E**). Colonies varied both in color (whitish, brown, deep green, and/or black) and texture (flat, fluffy, and/or wooly); sometimes *Alternaria* colonies grown on artificial media display typical patterns including different colors and textures. Within *A. alternata* complex, *alternata* is the most spread morphotype that has dark brown conidia (20 ± 10 μm in diameter), ovalellipsoidal shaped, with 4 ± 1 transverse septa; conidia are arranged in branched chains. Colonies belonging to morphotype *tenuissima* are greenish with white margins; their conidia are elongated with a long-tapered beak. *A. alternata* morphotype *limoniasperae* is less common but observed as pomegranate pathogen too; colonies are flat, light brown, and granulated with undulating edges, while conidia are long, ellipsoidal, and display 1–3 transverse septa. The second *Alternaria* species recorded on pomegranate fruit is *A. arborescens* [26] whose colonies are greenishgray or brown in color, characterized by a slow growth rate; conidia shape ranges from oval to ellipsoidal, with 1–4 transverse and 1–2 longitudinal septa. Being erect and straight, branched, bended, and geniculate, *A. burnsii* conidiophores are typical whereas conidia are ununiformly slight but featured by a short beak [30]. Due to morphological heterogeneity, molecular approach is useful to identify species and morphotypes, particularly it may be advantageous using OPA 1**–**3 or OPA 10**–**2 barcoding regions [31] and/or a multilocus approach as described by Aloi et al. [26].

#### *2.1.2 Gray mold*

Gray mold is among the main postharvest diseases of pomegranates all over the world [32–34]. As proved by Testempasis and colleagues [35], gray mold is caused by species belonging to *B. cinerea* complex as *B. cinerea s.s.*, *B. pseudocinerea,* and *B. cinerea* group *S*., being the former two the most spread etiological agents. The infection starts in the crown area showing small tan-colored spots, which rapidly spread to the whole fruit (**Figure 2A**-**C**). Developing, spots became darker and softer until causing rind collapse; finally, gray fluffy mycelium and black sclerotia grow (**Figure 2D**). Internally, softening and browning of the arils and the development of gray mycelium feature this infection. Aiming to control postharvest losses, nesting among contiguous fruit is important because this significantly enhances pathogen diffusion. Early, colonies are whitish (**Figure 2E**, **F**), then become brownish-gray, and finally covered by sclerotia circle-arranged. Lemon-like conidia measure 7.7 ± 2.4 × 6.8 ± 2.5 μm on average, while sclerotia are 2.9 ± 1.5 × 2.1 ± 0.6 mm. Morphological identification within the species complex is not possible, therefore a duplex PCR assay to evaluate indels in the *mrr1* gene is the optimal solution; suggested primer pairs are BcinN-in-F/ BcinN-in-R and Mrr1-spez-F/Mrr1-spez-R [35].

#### *2.1.3 Coniella rot*

Considering symptoms, especially at early stage, Coniella fruit rot resembles gray mold. Hence, specific features must be considered. Coniella rot is caused by *Coniella granati* which is synonymized with *Pilidiella granati*; this host-specific fungus is well-known since the end of the nineteenth century when it was isolated *Pomegranate: Postharvest Fungal Diseases and Control DOI: http://dx.doi.org/10.5772/intechopen.109665*

#### **Figure 2.**

*Gray mold caused by* Botrytis cinerea*. (A) Early stage of rot. (B) Close-up image of sporulated gray mycelium, (C) infected stamens, and (D) black sclerotia. (E)* B. cinerea *young colony on PDA. (F) Nesting secondary infection among fruit [6].*

in Italy [36]. Nowadays, it is worldwide spread from Spain and Greece till Tunisia and South Africa causing both shoot blight and canker disease and the abovementioned crown rot [37–40]. Crown rot symptoms consist of small circular rind spots, spread around the calyx area; these rapidly broaden and make lesions softer and darker till reaching brown color (**Figure 3A**). At maturity, a thin whitish mycelium with spherical pycnidia, ranging between dark brown and black in color, covers the lesions. Internally fruit decay is soft and brown and involves arils too (**Figure 3B**,**C**). Usually, due to softness, decayed part of each fruit splits. PDA colony ranges between white and creamy in color, texture is leathery and covered by abundant dark pycnidia (110 ± 30 μm in diameter, **Figure 3D**) with thin membranous walls. In addition, hyphae are septate and conidia (13.75 ± 3.750 × 3.5 ± 1.5 μm) are unicellular and hyaline, ellipsoid to fusiform in shape, straight or slightly curved. Even though morphological identification is relevant, molecular confirmation based on Internal Transcribed Spacer ITS5/ITS4 is useful [6].

#### *2.1.4 Anthracnose*

Anthracnose is increasingly widespread, especially in tropical and subtropical areas where rainfall and damp wind could enhance its dissemination when temperatures increase [41]; although climate change is favoring its diffusion in the Mediterranean region, it is retained as a minor disease. Species belonging to *Colletotrichum* species-complex are the etiological agents; usually, pomegranate fruit symptoms are superimposable among different species displaying typical anthracnose (**Figure 4A**). Both species-complex and species are differently distributed in the world, indeed in the southeastern United States *C. theobromicola*, *C. siamense*,

#### **Figure 3.**

*Coniella granati rot. (A) External symptoms. (B) View of internal decay. (C) Close-up of the soft rot. (D) Colony on PDA plate [6].*

*C. gloeosporioides*, *C. nymphaeae*, *C. fiorinae*, and *C. simmondsii* are common postharvest pathogens of pomegranates [42]. Similarly, in Brazil, *C. theobromicola*, *C. siamense*, *C. gloeosporioides*, and *C. tropicale* are recorded [43, 44], instead in the old countries most widespread species belong to *C. acutatum* complex and *C. gloeosporioides* complex. For instance, *C. gloeosporioides* is recorded in Turkey and in Albania [44] and *C. acutatum* is registered in Italy [6]. Generally, anthracnose causes the typical soft sunken lesions, which merge as they grow; progressively, white mycelium develops onto and lesions become circular, concentric, and brown with darker spots (**Figure 4B**). On PDA, strains of *C. acutatum* appear fluffy, early white with a reverse ranging from salmon till grayish color, then peachy-pink with pinkish-salmon conidial masses (**Figure 4C**). On the other hand, mycelium of *C. gloeosporioides* displays a texture ranging from faintly aerial to dense cottony; color of colonies ranges from white to pale-olivaceous reaching gray. Acervuli are abundant, small dark-based with sprinkled setae. Meanly, conidia of *C. acutatum s.s.* and *C. gloeosporioides s.s.* measure 11.3 ± 2.8 × 4.2 ± 1.1 μm and 12 ± 2.9 × 4.9 ± 2.1 μm, respectively, although, generally, the size is host specific (**Figure 4C**). Both chief species are one-celled, but the first has elliptical-fusiform conidia, instead the last shows oval to oblong, end-pointed conidia. Being species-complex, morphological features are not enough to identify each species within the complex, therefore is needed a molecular multilocus approach according to species-complex they belong [45].

*Pomegranate: Postharvest Fungal Diseases and Control DOI: http://dx.doi.org/10.5772/intechopen.109665*

#### **Figure 4.**

*Anthracnose caused by* Colletotrichum acutatum s.s. *Artificial (A) and natural (B) infections. (C)*  Colletotrichum acutatum s.s. *colony grown on PDA plate and (D) conidia [6].*

#### **2.2 Wound infections**

#### *2.2.1 Blue-green mold*

The main fungal group involved in pomegranate wound infections belong to *Penicillium sensu lato* (*s.l.*) that includes *Penicillium s.s*. and *Talaromyces* genera; within this last genus, just *T. albobivertillius* species is recorded, which results spread both in Italy and China [46, 47]. In *Penicillium s.s.* group, the most abundant species are *P. glabrum*, *P. adametzioides*, and *P. brevicompactum.* Minor species belonging to this genus are *P. jhonkrugii*, *P. pagulum*, and *P. citrinum*. Among these, being not pathogenic for pomegranate fruit, *P. brevicompactum* and *P. jhonkrugii* are not relevant; however, producing cytotoxic compounds named brevianamide A and mycophenolic acid [48], *P. brevicompactum* is putatively hazardous for human health. *P. glabrum* has been reported with high incidence also in Greece, Spain, Uzbekistan, and Slovak Republic [37, 49–52], while *P. adametzioides* has been isolated in Israel [53] too. Although in Italy is believed a minor species, *P. citrinum* (synonymized with *P. implicatum*) is spread in Slovak Republic [51] and Pakistan [53]; being widespread and a citrinin producer [54], its presence is relevant; indeed, citrinin is a nephrotoxic mycotoxin [55, 56]. According to most up-date literature, other *Penicillium* species involved as postharvest pathogens of pomegranate fruit are *P. expansum*, *P. sclerotiorum*, and *P. minioluteum*, the last being synonymized with *Talaromyces minioluteus* and re-arranged in *T. minioluteus*-complex, that includes eight species [57].

Usually, lesions due to these fungi appear as brownish and circular necrotic areas, which became darker, deeper, and occasionally irregularly shaped until reach and infect arils. Finally, blue-green sporification grow inside or onto infected pomegranates. Infected stamens can serve as source of secondary inoculum increasing fungal diffusion (**Figure 5A**-**D**). Cultured colonies of *Penicillium s.l.* display different textures, from powdery to crustose or velvety; in general, they are blue-green colored and rounded by concentric whitish margins of different thicknesses and/or roughness. The reverse side of each colony has different colors according to pigments and/ or metabolites produced, although, often, shades range between white-yellow or red-brownish. Due to species-specificity of produced metabolites, the metabolomic profile is useful for species identification. Regarding micromorphology, features vary based on species, although typical brush-like conidiophores have spherical and unicellular conidia and are arranged as unbranching chains on the top of the phialides. Conidia diameter, which meanly ranges between 2.5 and 5 μm, and wall ornamentation should be useful for species identification. However, morphological identification of *Penicillium s.l.* species is complex; indeed, molecular confirmation by PCR is needed. Bt2a/Bt2b primer pair is used to identify *Penicillium s.l.* species amplifying a portion of the β-tubulin gene [48].
