Occurrence of Alternaria alternata and A. tenuissima Causing Black Rot in Cherry Fruits (Prunus avium) in Central Chile

Abstract

During the harvest of 2020 and 2021, sweet cherry (Prunus avium) fruit showed a firm rot with irregular pale to dark brown lesions on the fruit surface, with green to light brown fungal growth resembling Alternaria-like infection (Simmons, 2007). Diseased cherries (n= 80 fruit) were collected at harvest in mature (over 10-year-old) commercial orchards of cherry tree varieties Lapins, Regina, Santina, Skeena, and Sweetheart planted in four localities of the regions O´Higgins (33°59´ S, 70°42´W; San Francisco de Mostazal and Graneros) and Maule (35°00’S, 71°23´W; Curicó and Sagrada Familia), Central Chile. The incidence of black rot was 1.9 and 3.2% in O´Higgins and Maule region, respectively, and it was increased to up to 5% during cold storage. The fruit collected previously, were transported to the lab, and surface disinfected in 75% ethanol for 15 s, and rinsed in sterile water. Internal pieces from the junction of diseased and healthy tissues of fruits were placed on potato dextrose agar (PDA, 2%) for 7 days at 20°C. Forty-two isolates of Alternaria-like (Simmons, 2007) were recovered consistently from pure cultures taking hyphal tips from 7 days old cultures. On PDA, 28 isolates (group A) were characterized by cottony, white-gray to green colonies and conidial chains (4 to 10 conidia) with secondary chains (1 to 5 conidia) branching on the conidiophore. Conidia were ovate to obclavate (mean 22.8 ± 5.1 x 8.8 ± 1.5 μm; n=40) with 3 to 7 transepta and 1 longisepta. The remaining 14 isolates (group B) were characterized by cottony, olive-green to olive-brown colonies following a ring pattern of growth and white margins, with conidial chains (4 to 14 conidia) and uncommon secondary chains (1 to 4 conidia). Conidia were obpyriform to ovate, light brown to brown with a cylindrical short beak at the tip (mean 24.7 ± 5.9 × 11.2 ± 1.3 μm; n=40) with 2 to 4 transepta, and 0 to 2 longisepta. Two representative isolates of group A (Sant-02-2020 and Bing-03-2020) and group B (Sant-26-2021 and Skeen-43-2021) were amplified for the Alternaria major allergen (Alt a1), plasma membrane ATPase (ATP), and calmodulin (Cal) loci following the protocols described by Hong et al. (2005) and Lawrence et al. (2013). A MegaBlast search of sequences of group A (GenBank nos. OR267293- OR267294, OR258001- OR258002, and OR267297- OR267298, for Alt a1, ATP, and Cal, respectively) showed 100% similarity to strains UCD10529 and UCD10539 of A. alternata, and group B (GenBank nos. OR267295- OR267296, OR258003- OR258004, and OR258005- OR258006, for Alt a1, ATP, and Cal, respectively) showed 100% similarity to strains EGS 34-015 and A30 of A. tenuissima. Combined phylogenetic analysis using MEGA X clustered isolates Sant-02-2020 and Bing-03-2020, and Sant-26-2021 and Skeen-43-2021 with ex-type of A. alternata and A. tenuissima, respectively. Pathogenicity tests were conducted using isolates of A. alternata (Sant-02-2020; Bing-03-2020) and A. tenuissima (Sant-26-2021; Skeen-43-2021). Detached ripe cherry fruit var. Sweetheart (n=40 fruits/isolate) and Regina (n=40 fruits/isolate) were surfaces disinfested (75% ethanol, 30 s), wounded in the middle with a sterile needle (2 mm in depth), and inoculated with 20 μL of conidial suspension (106 conidia/mL). An equal number of healthy cherries (n=40 fruits) treated with sterile water were used as controls. The experiment was repeated once. All inoculated fruit incubated for 7 days at 22°C, developed between 13 ± 2.7 to 23 ± 2.5 mm and 14.1 ± 1.1 to 19 ± 3.6 mm in lesion diameter for A. alternata and A. tenuissima isolates, respectively. Koch´s postulates were fulfilled by 100% reisolation of the causal pathogen from inoculated fruit, and molecular identification of A. alternata and A. tenuissima isolates. Previously, A. alternata has been described as causing rots on cherries in Chile (Acuña 2010), and China (Zhao and Liu, 2012; Ahmad et al., 2020). To our knowledge, this is the first occurrence of cherry black rot caused by A. alternata and A. tenuissima in Central Chile. Epidemiological studies are necessary to develop integrated management of cherry black rot in Central Chile.

Molecular Characterization and Pathogenicity of Alternaria spp. Associated with Black Rot of Sweet Cherries in Italy

“…Keissl. was recently reported on cherries in Chile [9] and in China [8,10]. A. alternata is also associated with brown to black spots on cherry leaves [24,25].…”

“…Sweet cherries are highly perishable fruit, with a shelf life of 7-14 days [3] and they are susceptible, during cold storage, to decay caused by postharvest pathogens, including Monilinia spp., agent of brown rot; Botrytis cinerea, agent of gray mold; Penicillium expansum, agent of blue mold; and Rhizopus spp., agent of soft rot [4][5][6][7]. Cherry production could be threatened also by black rot caused by Alternaria species, which is emerging as a major fungal disease [8][9][10]. Symptoms of black rot of Alternaria appear as dark brown to black patches on the outer surface of fruits.…”

Occurrence and Characterization of Sclerotinia sclerotiorum Causing Fruit Rot on Sweet Cherry in Southern China

“…The two isolates caused the same symptoms when inoculated on young fruits of sweet cherry as that in the field, labeled as ScSs1 and ScSs2 (Figure 1D,E). [14,[21][22][23][24]. B. cinerea and M. fructicola were also identified to cause fruit rot in cherries in China [25,26].…”

“…They occur throughout the whole fruit development period, from the young fruit stage to the post-harvest stage. Several pathogenic fungi have been reported to cause fruit rot in California, Oregon, Maryland, of the USA, and Santiago, O’Higgins, Maule, of Chile, including Monilinia fructicola , Botrytis cinerea , Sclerotinia sclerotiorum , and Alternaria alternata , which are all important pathogens in agricultural production [ 14 , 21 , 22 , 23 , 24 ]. B. cinerea and M. fructicola were also identified to cause fruit rot in cherries in China [ 25 , 26 ].…”

Characterization and toxicological potential of Alternaria alternata associated with post-harvest fruit rot of Prunus avium in China

“…Alternaria alternata and Alternaria tenuissima in central Chile (Cancino et al, 2023) and Alternaria arborescens species complex (AASC) and A. alternata in Italy (Waqas et al, 2023) have been identified as the causal agents of black rot disease in sweet cherries based on the morphological and molecular analyses. These causal agents significantly contribute to yield losses.…”